Mouse having an engineered immunoglobulin lambda light chain

ABSTRACT

Non-human animals (and/or non-human cells) and methods of using the same are provided, which non-human animals (and/or non-human cells) have a genome comprising human antibody-encoding sequences (i.e., immunoglobulin genes). Non-human animals described herein express antibodies that contain immunoglobulin (Ig) light chains characterized by the presence of human Vλ domains. Non-human animals provided herein are, in some embodiments, characterized by expression of antibodies that contain human Vλ light chains that are encoded by human Igλ light chain-encoding sequences inserted into an endogenous Igκ light chain locus of said non-human animals. Methods for producing antibodies from non-human animals are also provided, which antibodies contain human variable regions and mouse constant regions.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/594,944, filed Dec. 5, 2017; U.S. Provisional Application No.62/594,946, filed Dec. 5, 2017; U.S. Provisional Application No.62/609,241, filed Dec. 21, 2017; and U.S. Provisional Application No.62/609,251, filed Dec. 21, 2017; each of which is incorporated herein byreference.

SEQUENCE LISTING

The instant application contains a Sequence Listing, which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. The ASCII copy, created Jul. 30, 2020, isnamed 2010794-1443_SL.txt, and is 29,403 bytes in size.

BACKGROUND

Human antibodies are the most rapidly growing class of therapeutics. Ofthe technologies that are currently used for their production, thedevelopment of genetically engineered animals (e.g., rodents) engineeredwith genetic material encoding human antibodies, in whole or in part,has revolutionized the field of human therapeutic monoclonal antibodiesfor the treatment of various diseases. Still, development of improved invivo systems for generating human monoclonal antibodies that maximizehuman antibody repertoires in host genetically engineered animals isneeded.

SUMMARY

In some embodiments, the present disclosure provides a rodent, whosegermline genome includes:

an engineered endogenous immunoglobulin κ light chain locus including:

-   -   (a) one or more human Vλ gene segments,    -   (b) one or more human Jλ gene segments, and    -   (c) one or more Cλ genes,

where the one or more human Vλ gene segments and the one or more humanJλ gene segments are operably linked to the one or more Cλ genes, andwhere the rodent lacks a rodent Cκ gene at the engineered endogenousimmunoglobulin κ locus.

In some embodiments, one or more Cλ genes is a Cλ gene. In someembodiments, a Cλ gene is or includes a rodent Cλ gene. In someembodiments, a rodent Cλ gene has a sequence that is at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, or at least 99%identical to a mouse Cλ1, mouse Cλ2 or a mouse Cλ3 gene. In someembodiments, a rodent Cλ gene is or includes a mouse Cλ1 gene. In someembodiments, a rodent Cλ gene is or includes a rat Cλ gene. In someembodiments, a rat Cλ gene has a sequence that is at least at least 80%,at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%identical to a rat Cλ1, rat Cλ2, rat Cλ3 or a rat Cλ4 gene.

In some embodiments, one or more human Vλ gene segments and one or morehuman Jλ gene segments are in place of one or more rodent Vκ genesegments, one or more rodent Jκ gene segments, or any combinationthereof. In some embodiments, one or more human Vλ gene segments and oneor more human Jλ gene segments replace one or more rodent Vκ genesegments, one or more rodent Jκ gene segments, or any combinationthereof. In some embodiments, one or more human Vλ gene segments and oneor more human Jλ gene segments replace all functional rodent Vκ genesegments and/or all functional rodent Jκ gene segments.

In some embodiments, one or more human Vλ gene segments include Vλ4-69,Vλ8-61, Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46,Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25,Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10,Vλ3-9, Vλ2-8, Vλ4-3, Vλ3-1, or any combination thereof. In someembodiments, one or more human Vλ gene segments include Vλ4-69, Vλ8-61,Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45,Vλ1-44, Vλ7-43, Vλ1- 40, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19,Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, Vλ3-1, orany combination thereof. In some embodiments, one or more human Vλ genesegments include Vλ4-69, Vλ8-61, Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52,Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39,Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16,Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, and Vλ3-1.

In some embodiments, one or more human Jλ gene segments include Jλ1,Jλ2, Jλ3, Jλ6, Jλ7, or any combination thereof. In some embodiments, oneor more human Jλ gene segments include Jλ1, Jλ2, Jλ3, Jλ6, and Jλ7.

In some embodiments, an engineered endogenous immunoglobulin κ lightchain locus includes one or more human Vλ non-coding sequences, each ofwhich is adjacent to at least one of the one or more human Vλ genesegments, where the one or more human Vλ non-coding sequences naturallyappears adjacent to a human Vλ gene segment in an endogenous humanimmunoglobulin λ light chain locus. For example, referring to FIG. 20, afirst exemplary endogenous human Vλ non-coding sequence naturallyappears adjacent (and 3′) to a Vλ3-12 gene segment in an endogenoushuman immunoglobulin λ light chain locus. An engineered endogenousimmunoglobulin κ light chain locus including the first exemplaryendogenous human Vλ non-coding sequence could include that non-codingsequence at a position that is adjacent (and preferably 3′) to a Vλ3-12gene segment in the engineered endogenous immunoglobulin κ light chainlocus. An engineered endogenous immunoglobulin κ light chain locusincluding the first exemplary endogenous human Vλ non-coding sequencecould also include that non-coding sequence at a position that isadjacent (and preferably 5′) to a Vλ2-11 gene segment in the engineeredendogenous immunoglobulin κ light chain locus. In some instances, anengineered endogenous immunoglobulin κ light chain locus including thefirst exemplary endogenous human Vλ non-coding sequence could alsoinclude that non-coding sequence at a position that is adjacent (andpreferably 3′) to a Vλ3-12 gene segment and adjacent (and preferably 5′)to a Vλ2-11 gene segment in the engineered endogenous immunoglobulin κlight chain locus. In some embodiments, each of the one or more human Vλnon-coding sequences is or includes an intron.

In some embodiments, an engineered endogenous immunoglobulin κ lightchain locus includes one or more human Jλ non-coding sequences, each ofwhich is adjacent to at least one of the one or more human Jλ genesegments, where the one or more human Jλ non-coding sequences naturallyappears adjacent to a human Jλ gene segment in an endogenous humanimmunoglobulin λ light chain locus. In some embodiments, each of the oneor more human Jλ non-coding sequences is or includes an intron. In someembodiments, an engineered endogenous immunoglobulin κ light chain locusincludes one or more human Jκ non-coding sequences, each of which isadjacent to at least one of the one or more human Jλ gene segment, wherethe one or more human Jκ non-coding sequences naturally appears adjacentto a human Jκ gene segment in an endogenous human immunoglobulin κ lightchain locus. For example, referring to FIG. 21, a first exemplaryendogenous human Jκ non-coding sequence naturally appears in anendogenous human immunoglobulin κ light chain locus. An engineeredendogenous immunoglobulin κ light chain locus including the firstexemplary endogenous human Jκ non-coding sequence could be a non-codingsequence at a position that is adjacent to a Jλ gene segment (e.g., Jλ1,Jλ2, Jλ3, Jλ6 or Jλ7) in the engineered endogenous immunoglobulin κlight chain locus. In some embodiments, each of the one or more human Jκnon-coding sequences is or includes an intron.

In some embodiments, an engineered endogenous immunoglobulin κ lightchain locus includes one or more human Vλ non-coding sequences, whereeach of the one or more human Vλ non-coding sequences is adjacent to theVλ4-69, Vλ8-61, Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47,Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27,Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11,Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, or Vλ3-1 in the engineered endogenousimmunoglobulin κ light chain locus, and where each of the one or morehuman Vλ non-coding sequences naturally appear adjacent to a Vλ4-69,Vλ8-61, Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46,Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25,Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10,Vλ3-9, Vλ2-8, Vλ4-3, or Vλ3-1 of an endogenous human immunoglobulin λlight chain locus. In some embodiments, an engineered endogenousimmunoglobulin κ light chain locus includes one or more human Jλnon-coding sequences, where each of the one or more human Jλ non-codingsequences is adjacent to the Jλ1, Jλ2, Jλ3, Jλ6 or Jλ7 in the engineeredendogenous immunoglobulin κ light chain locus, and where each of the oneor more human Jλ non-coding sequences naturally appear adjacent to aJλ1, Jλ2, Jλ3, Jλ6 or Jλ7 of an endogenous human immunoglobulin λ lightchain locus. In some embodiments, an engineered endogenousimmunoglobulin κ light chain locus includes one or more human Jκnon-coding sequences, where each of the one or more human Jκ non-codingsequences is adjacent to the Jλ1, Jλ2, Jλ3, Jλ6 or Jλ7 in the engineeredendogenous immunoglobulin κ light chain locus, and where each of the oneor more human Jκ non-coding sequences naturally appear adjacent to aJκ1, Jκ2, Jκ3, Jκ4, or Jκ5 of an endogenous human immunoglobulin κ lightchain locus.

In some embodiments, an engineered endogenous immunoglobulin κ lightchain locus includes a κ light chain non-coding sequence between the oneor more human Vλ gene segments and the one or more human Jλ genesegments. In some embodiments, a κ light chain non-coding sequence is ahuman κ light chain non-coding sequence. In some embodiments, a human κlight chain non-coding sequence has a sequence that naturally appearsbetween a human Vκ4-1 gene segment and a human Jκ1 gene segment in anendogenous human immunoglobulin κ light chain locus.

In some embodiments, a rodent described herein is homozygous for anengineered endogenous immunoglobulin κ light chain locus. In someembodiments, a rodent described herein is heterozygous for an engineeredendogenous immunoglobulin κ light chain locus. In some embodiments, thegermline genome of a rodent includes a second engineered endogenousimmunoglobulin κ light chain locus that includes:

(a) one or more human Vκ gene segments, and

(b) one or more human Jκ gene segments,

where the one or more human Vκ gene segments and the one or more humanJκ gene segments are operably linked to a Cκ gene.

In some embodiments, the genome of the rodent further includes a nucleicacid sequence encoding an exogenous terminal deoxynucleotidyltransferase(TdT) operably linked to a transcriptional control element. In someembodiments, the transcriptional control element includes a RAG1transcriptional control element, a RAG2 transcriptional control element,an immunoglobulin heavy chain transcriptional control element, animmunoglobulin κ light chain transcriptional control element, animmunoglobulin λ light chain transcriptional control element, or anycombination thereof. In some embodiments, the nucleic acid sequenceencoding an exogenous TdT is located at an immunoglobulin κ light chainlocus, an immunoglobulin λ light chain locus, an immunoglobulin heavychain locus, a RAG1 locus, or a RAG2 locus. In some embodiments, a TdTis a human TdT. In some embodiments, a TdT is a short isoform of TdT(TdTS).

In some embodiments, a rodent described herein includes a nucleic acidsequence encoding an exogenous terminal deoxynucleotidyltransferase(TdT) operably linked to a transcriptional control element in itsgermline genome and exhibits light chains (e.g., expresses light chainvariable domains including) with at least a 1.2-fold, at least a1.5-fold, at least a 1.75-fold, at least a 2-fold, at least a 3-fold, atleast a 4-fold, or a least a 5-fold increase in junctional diversityover a comparable mouse (e.g., littermate) that does not include anexogenous terminal deoxynucleotidyltransferase (TdT) operably linked toa transcriptional control element in its germline genome. In someembodiments, junctional diversity is measured by number of uniqueCDR3/10,000 reads.

In some embodiments, a rodent described herein includes a nucleic acidsequence encoding an exogenous terminal deoxynucleotidyltransferase(TdT) operably linked to a transcriptional control element in itsgermline genome and at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 55%, at least 60%, at least65% of light chains (e.g., lambda and/or kappa light chains) produced bythe rodent exhibit non-template additions.

In some embodiments, a germline genome of a rodent described hereinincludes: an engineered endogenous immunoglobulin heavy chain locus,including:

-   -   (a) one or more human V_(H) gene segments,    -   (b) one or more human D_(H) gene segments, and    -   (c) one or more human J_(H) gene segments,

where the one or more human V_(H) gene segments, the one or more humanD_(H) gene segments, and the one or more human J_(H) gene segments areoperably linked to a rodent immunoglobulin heavy chain constant regionat the engineered endogenous immunoglobulin heavy chain locus.

In some embodiments, one or more human V_(H) gene segments, one or morehuman D_(H) gene segments, and one or more human J_(H) gene segments arein place of one or more rodent V_(H) gene segments, one or more rodentD_(H) gene segments, one or more rodent J_(H) gene segments, or acombination thereof. In some embodiment, one or more human V_(H) genesegments, one or more human D_(H) gene segments, and one or more humanJ_(H) gene segments replace one or more rodent V_(H) gene segments, oneor more rodent D_(H) gene segments, one or more rodent J_(H) genesegments, or any combination thereof.

In some embodiments, one or more human V_(H) gene segments includeV_(H)3-74, V_(H)3-73, V_(H)3-72, V_(H)2-70, V_(H)1-69, V_(H)3-66,V_(H)3-64, V_(H)4-61, V_(H)4-59, V_(H)1-58, V_(H)3-53, V_(H)5-51,V_(H)3-49, V_(H)3-48, V_(H)1-46, V_(H)1-45, V_(H)3-43, V_(H)4-39,V_(H)4-34, V_(H)3-33, V_(H)4-31, V_(H)3-30, V_(H)4-28, V_(H)2-26,V_(H)1-24, V_(H)3-23, V_(H)3-21, V_(H)3-20, V_(H)1-18, V_(H)3-15,V_(H)3-13, V_(H)3-11, V_(H)3-9, V_(H)1- 8, V_(H)3-7, V_(H)2-5,V_(H)7-4-1, V_(H)4-4, V_(H)1-3, V_(H)1-2, V_(H)6-1, or any combinationthereof. In some embodiments, one or more human V_(H) gene segmentsinclude V_(H)3-74, V_(H)3-73, V_(H)3-72, V_(H)2-70, V_(H)1-69,V_(H)3-66, V_(H)3-64, V_(H)4-61, V_(H)4-59, V_(H)1-58, V_(H)3-53,V_(H)5-51, V_(H)3-49, V_(H)3-48, V_(H)1-46, V_(H)1-45, V_(H)3-43,V_(H)4-39, V_(H)4-34, V_(H)3-33, V_(H)4-31, V_(H)3-30, V_(H)4-28,V_(H)2-26, V_(H)1-24, V_(H)3-23, V_(H)3-21, V_(H)3-20, V_(H)1-18,V_(H)3-15, V_(H)3-13, V_(H)3-11, V_(H)3-9, V_(H)1-8, V_(H)3-7, V_(H)2-5,V_(H)7-4-1, V_(H)4-4, V_(H)1-3, V_(H)1-2, and V_(H)6-1.

In some embodiments, one or more human D_(H) gene segments includeD_(H)1-1, D_(H)2-2, D_(H)3-3, D_(H)4-4, D_(H)5-5, D_(H)6-6, D_(H)1-7,D_(H)2-8, D_(H)3-9, D_(H)3-10, D_(H)5-12, D_(H)6-13, D_(H)2-15,D_(H)3-16, D_(H)4-17, D_(H)6-19, D_(H)1-20, D_(H)2-21, D_(H)3-22,D_(H)6-25, D_(H)1-26, D_(H)7-27, or any combination thereof. In someembodiments, one or more human D_(H) gene segments include D_(H)1-1,D_(H)2-2, D_(H)3-3, D_(H)4-4, D_(H)5-5, D_(H)6-6, D_(H)1-7, D_(H)2-8,D_(H)3-9, D_(H)3-10, D_(H)5-12, D_(H)6-13, D_(H)2-15, D_(H)3-16,D_(H)4-17, D_(H)6-19, D_(H)1-20, D_(H)2-21, D_(H)3-22, D_(H)6-25,D_(H)1-26, and D_(H)7-27.

In some embodiments, one or more human J_(H) gene segments includeJ_(H)1, J_(H)2, J_(H)3, J_(H)4, J_(H)5, J_(H)6, or any combinationthereof. In some embodiments, one or more human J_(H) gene segmentsinclude J_(H)1, J_(H)2, J_(H)3, J_(H)4, J_(H)5, and J_(H)6.

In some embodiments, an engineered endogenous immunoglobulin heavy chainlocus includes one or more human V_(H) non-coding sequences, each ofwhich is adjacent to at least one of the one or more human V_(H) genesegments, where each of the one or more V_(H) non-coding sequencesnaturally appears adjacent to a human V_(H) gene segment in anendogenous human immunoglobulin heavy chain locus. In some embodiments,each of the one or more human V_(H) non-coding sequences is or includesan intron. In some embodiments, an engineered endogenous immunoglobulinheavy chain locus includes one or more human D_(H) non-coding sequences,each of which is adjacent to at least one of the one or more human D_(H)gene segments, where each of the one or more D_(H) non-coding sequencesnaturally appears adjacent to a human D_(H) gene segment in anendogenous human immunoglobulin heavy chain locus. In some embodiments,each of the one or more human D_(H) non-coding sequences is or includesan intron. In some embodiments, an engineered endogenous immunoglobulinheavy chain locus includes one or more human J_(H) non-coding sequences,each of which is adjacent to at least one of the one or more human J_(H)gene segments, where each of the one or more J_(H) non-coding sequencesnaturally appears adjacent to a human J_(H) gene segment in anendogenous human immunoglobulin heavy chain locus. In some embodiments,each of the one or more human J_(H) non-coding sequences is or includesan intron.

In some embodiments, a rodent described herein is homozygous for anengineered endogenous immunoglobulin heavy chain locus.

In some embodiments, a rodent immunoglobulin heavy chain constant regionis an endogenous rodent immunoglobulin heavy chain constant region.

In some embodiments, endogenous Vλ gene segments, endogenous Jλ genesegments, and the endogenous Cλ genes are deleted in whole or in part.In some embodiments, a rodent described herein does not detectablyexpress endogenous immunoglobulin λ light chain variable domains. Insome embodiments, a rodent described herein does not detectably expressendogenous immunoglobulin κ light chain variable domains.

In some embodiments, an engineered endogenous immunoglobulin heavy chainlocus lacks a functional endogenous rodent Adam6 gene. In someembodiments, a germline genome of a rodent includes one or morenucleotide sequences encoding one or more rodent ADAM6 polypeptides,functional orthologs, functional homologs, or functional fragmentsthereof. In some embodiments, one or more rodent ADAM6 polypeptides,functional orthologs, functional homologs, or functional fragmentsthereof are expressed (e.g., in a cell of the male reproductive system,e.g., a testes cell).

In some embodiments, one or more nucleotide sequences encoding one ormore rodent ADAM6 polypeptides, functional orthologs, functionalhomologs, or functional fragments thereof are included on the samechromosome as the engineered endogenous immunoglobulin heavy chainlocus. In some embodiments, one or more nucleotide sequences encodingone or more rodent ADAM6 polypeptides, functional orthologs, functionalhomologs, or functional fragments thereof are included in the engineeredendogenous immunoglobulin heavy chain locus. In some embodiments, one ormore nucleotide sequences encoding one or more rodent ADAM6polypeptides, functional orthologs, functional homologs, or functionalfragments thereof are between a first human V_(H) gene segment and asecond human V_(H) gene segment. In some embodiments, a first humanV_(H) gene segment is V_(H)1-2 and a second human V_(H) gene segment isV_(H)6-1. In some embodiments, one or more nucleotide sequences encodingone or more rodent ADAM6 polypeptides, functional orthologs, functionalhomologs, or functional fragments thereof are in place of a human Adam6pseudogene. In some embodiments, one or more nucleotide sequencesencoding one or more rodent ADAM6 polypeptides, functional orthologs,functional homologs, or functional fragments thereof replace a humanAdam6 pseudogene. In some embodiments, one or more nucleotide sequencesencoding one or more rodent ADAM6 polypeptides, functional orthologs,functional homologs, or functional fragments thereof are between a humanV_(H) gene segment and a human D_(H) gene segment.

In some embodiments, a rodent described herein includes a population ofB cells that express antibodies, including immunoglobulin λ light chainsthat each include a human immunoglobulin λ light chain variable domain.In some embodiments, a human immunoglobulin λ light chain variabledomain is encoded by a rearranged human immunoglobulin λ light chainvariable region sequence including (i) one of the one or more human Vλgene segments or a somatically hypermutated variant thereof, and (ii)one of the one or more human Jλ gene segments or a somaticallyhypermutated variant thereof.

In some embodiments, a rodent described herein includes a population ofB cells that express antibodies, including immunoglobulin heavy chainsthat each include a human immunoglobulin heavy chain variable domain. Insome embodiments, a human immunoglobulin heavy chain variable domain isencoded by a rearranged human immunoglobulin heavy chain variable regionsequence including (i) one of the one or more human V_(H) gene segmentsor a somatically hypermutated variant thereof, (ii) one of the one ormore human D_(H) gene segments or a somatically hypermutated variantthereof, and (ii) one of the one or more human J_(H) gene segments or asomatically hypermutated variant thereof.

In some embodiments, a rodent described herein produces a population ofB cells in response to immunization with an antigen that includes one ormore epitopes. In some embodiments, a rodent produces a population of Bcells that express antibodies that bind (e.g., specifically bind) to oneor more epitopes of antigen of interest. In some embodiments, antibodiesexpressed by a population of B cells produced in response to an antigeninclude a heavy chain having a human heavy chain variable domain encodedby a human heavy chain variable region sequence and/or a lambda lightchain having a human lambda light chain variable domain encoded by ahuman lambda light chain variable region sequence as described herein.In some embodiments, antibodies expressed by a population of B cellsproduced in response to an antigen include a heavy chain having a humanheavy chain variable domain encoded by a human heavy chain variableregion sequence and/or a kappa light chain having a human kappa lightchain variable domain encoded by a human kappa light chain variableregion sequence as described herein.

In some embodiments, a rodent produces a population of B cells thatexpress antibodies that bind to one or more epitopes of antigen ofinterest, where antibodies expressed by the population of B cellsproduced in response to an antigen include: (i) a heavy chain having ahuman heavy chain variable domain encoded by a human heavy chainvariable region sequence, (ii) a lambda light chain having a humanlambda light chain variable domain encoded by a human lambda light chainvariable region sequence as described herein, (iii) a kappa light chainhaving a human kappa light chain variable domain encoded by a humankappa light chain variable region sequence as described herein, or (iv)any combination thereof.

In some embodiments, a human heavy chain variable region sequence, ahuman λ light chain variable region sequence, and/or a human κ lightchain variable region sequence as described herein is somaticallyhypermutated. In some embodiments, at least about 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% ofthe B cells in a population of B cells produced in response to anantigen include a human heavy chain variable region sequence, λ lightchain variable region sequence, and/or κ light chain variable regionsequence that is somatically hypermutated.

In some embodiments, a rodent described herein is a mouse or a rat.

In some embodiments, cells and/or tissues provided (e.g., isolated cellsand/or tissues) from a rodent are described herein. In some embodiments,provided cells and tissues include, for example, lymphoid tissue,splenocytes, B cells, stem cells and/or germ cells. In some embodiments,a provided cell is isolated. In some embodiments, an isolated cell is orincludes a pro B-cell, a pre-B cell, an immature B cell, a mature naïveB cell, an activated B cell, a memory B cell, a B lineage lymphocyte,and/or a plasma cell. In some embodiments, an isolated cell includes astem cell (e.g., an embryonic stem cell) and/or a germ cell (e.g.,sperm, oocyte).

In some embodiments, the present disclosure provides an isolated rodentcell, whose germline genome includes:

an engineered endogenous immunoglobulin κ light chain locus including:

-   -   (a) one or more human Vλ gene segments,    -   (b) one or more human Jλ gene segments, and    -   (c) a Cλ gene,

where the one or more human Vλ gene segments and the one or more humanJλ gene segments are operably linked to the Cλ gene.

In some embodiments, an isolated rodent cell described herein lacks arodent Cκ gene at the engineered endogenous immunoglobulin κ locus.

In some embodiments, an isolated rodent cell described herein is arodent embryonic stem (ES) cell.

In some embodiments, the present disclosure provides a rodent embryogenerated from a rodent ES cell described herein.

In some embodiments, the present disclosure provides an immortalizedcell generated from an isolated rodent cell described herein.

In some embodiments, the present disclosure provides a method of makinga rodent whose germline genome includes an engineered endogenousimmunoglobulin κ light chain locus, the method including the steps of:

(a) introducing one or more DNA fragments into the germline genome of arodent ES cell, where the one or more DNA fragments comprise:

-   -   (i) one or more human Vλ gene segments,    -   (ii) one or more human Jλ gene segments, and    -   (iii) one or more Cλ genes,    -   where the one or more human Vλ gene segments, the one or more        human Jλ gene segments, and the one or more Cλ genes are        introduced into the germline genome of the rodent ES cell at the        endogenous immunoglobulin κ light chain locus, and where the one        or more human Vλ gene segments, the one or more human Jλ gene        segments, and the one or more Cλ genes are operably linked; and

(b) generating a rodent using the rodent ES cell generated in (a).

In some embodiments, a method of making a rodent whose germline genomeincludes an engineered endogenous immunoglobulin κ light chain locus,includes the step of introducing a κ light chain non-coding sequenceinto the germline genome of the rodent ES cell so that the κ light chainnon-coding sequence is between the one or more human Vλ gene segmentsand the one or more human Jλ gene segments in the germline genome of therodent ES cell.

In some embodiments, the present disclosure provides a method of makinga rodent whose germline genome includes an engineered endogenousimmunoglobulin κ light chain locus, the method including the steps of:

engineering the endogenous immunoglobulin κ light chain locus in thegermline genome to include:

-   -   (a) one or more human Vλ gene segments,    -   (b) one or more human Jλ gene segments, and    -   (c) one or more Cλ genes,

where the one or more human Vλ gene segments and the one or more humanJλ gene segments are operably linked to the one or more Cλ genes, and

where the one or more Cλ genes are inserted in place of a rodent Cκ geneat the endogenous immunoglobulin κ locus.

In some embodiments, a Cλ gene replaces a rodent Cκ gene at theendogenous immunoglobulin κ locus.

In some embodiments, one or more human Vλ gene segments include Vλ5-52,Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39,Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18,Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, Vλ3-1, orany combination thereof. In some embodiments, one or more human Vλ genesegments include Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44,Vλ7-43, Vλ1-40, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18,Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, and Vλ3-1.In some embodiments, one or more human Vλ gene segments include Vλ5-52,Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39,Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18,Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, and Vλ3-1.

In some embodiments, one or more human Jλ gene segments includes Jλ1,Jλ2, Jλ3, Jλ6, Jλ7, or any combination thereof. In some embodiments, oneor more human Jλ gene segments includes Jλ1, Jλ2, Jλ3, Jλ6, and Jλ7.

In some embodiments, an engineered endogenous immunoglobulin κ lightchain locus includes one or more human Vλ non-coding sequences, each ofwhich is adjacent to at least one of the one or more human Vλ genesegments, where the one or more human Vλ non-coding sequences naturallyappears adjacent to a human Vλ gene segment in an endogenous humanimmunoglobulin λ light chain locus. In some embodiments, each of the oneor more human Vλ non-coding sequences is or includes an intron. In someembodiments, an engineered endogenous immunoglobulin κ light chain locusincludes one or more human Jλ non-coding sequences, each of which isadjacent to at least one of the one or more human Jλ gene segment, wherethe one or more human Jλ non-coding sequences naturally appears adjacentto a human Jλ gene segment in an endogenous human immunoglobulin λ lightchain locus. In some embodiments, each of the one or more human Jλnon-coding sequences is or includes an intron. In some embodiments, anengineered endogenous immunoglobulin κ light chain locus includes one ormore human Jκ non-coding sequences, each of which is adjacent to atleast one of the one or more human Jλ gene segment, where the one ormore human Jκ non-coding sequences naturally appears adjacent to a humanJκ gene segment in an endogenous human immunoglobulin κ light chainlocus. In some embodiments, each of the one or more human Jκ non-codingsequences is or includes an intron.

In some embodiments, a Cλ gene is or includes a rodent Cλ gene. In someembodiments, a rodent Cλ gene has a sequence that is at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, or at least 99%identical to a mouse Cλ1, mouse Cλ2 or a mouse Cλ3 gene. In someembodiments, a rodent Cλ gene is or includes a mouse Cλ1 gene. In someembodiments, a rodent Cλ gene is or includes a rat Cλ gene. In someembodiments, a rat Cλ gene has a sequence that is at least at least 80%,at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%identical to a rat Cλ1, rat Cλ2, rat Cλ3 or a rat Cλ4 gene.

In some embodiments, one or more DNA fragments include at least oneselection marker. In some embodiments, one or more DNA fragments includeat least one site-specific recombination site.

In some embodiments, the germline genome of a rodent includes:

an engineered endogenous immunoglobulin heavy chain locus, including:

(a) one or more human V_(H) gene segments,

(b) one or more human D_(H) gene segments, and

(c) one or more human J_(H) gene segments,

where the one or more human V_(H) gene segments, the one or more humanD_(H) gene segments, and the one or more human J_(H) gene segments areoperably linked to a rodent immunoglobulin heavy chain constant region.

In some embodiments, the step of engineering the endogenousimmunoglobulin κ light chain locus in the germline genome is carried outin a rodent ES cell whose germline genome includes an engineeredendogenous immunoglobulin heavy chain locus including one or more humanV_(H) gene segments, one or more human D_(H) gene segments, and one ormore human J_(H) gene segments operably linked to a rodentimmunoglobulin heavy chain constant region.

In some embodiments, an engineered endogenous immunoglobulin heavy chainlocus includes one or more human V_(H) non-coding sequences, each ofwhich is adjacent to at least one of the one or more human V_(H) genesegments, where each of the one or more human V_(H) non-coding sequencesnaturally appears adjacent to a human V_(H) gene segment in anendogenous human immunoglobulin heavy chain locus. In some embodiments,each of the one or more human V_(H) non-coding sequences is or includesan intron. In some embodiments, an engineered endogenous immunoglobulinheavy chain locus includes one or more human D_(H) non-coding sequences,each of which is adjacent to at least one of the one or more human D_(H)gene segments, where each of the one or more D_(H) non-coding sequencesnaturally appears adjacent to a human D_(H) gene segment in anendogenous human immunoglobulin heavy chain locus. In some embodiments,each of the one or more human D_(H) non-coding sequences is or includesan intron. In some embodiments, an engineered endogenous immunoglobulinheavy chain locus includes one or more human J_(H) non-coding sequences,each of which is adjacent to at least one of the one or more human J_(H)gene segments, where each of the one or more J_(H) non-coding sequencesnaturally appears adjacent to a human J_(H) gene segment in anendogenous human immunoglobulin heavy chain locus. In some embodiments,each of the one or more human J_(H) non-coding sequences is or includesan intron.

In some embodiments, the present disclosure provides a method ofproducing an antibody in a rodent, the method including the steps of:

(i) immunizing a rodent with an antigen of interest,

where the rodent has a germline genome including:

-   -   an engineered endogenous immunoglobulin κ light chain locus,        including:        -   (a) one or more human Vλ gene segments,        -   (b) one or more human Jλ gene segments, and        -   (c) one or more Cλ genes,    -   where the one or more human Vλ gene segments and the one or more        human Jλ gene segments are operably linked to the Cλ gene, and    -   where the one or more Cλ genes are in the place of a rodent Cκ        gene at the engineered endogenous immunoglobulin κ locus;

maintaining the rodent under conditions sufficient for the rodent toproduce an immune response to the antigen of interest; and

recovering an antibody that binds the antigen of interest from therodent, a cell of the rodent, or a cell derived from a cell of therodent.

In some embodiments, in response to the step of immunizing, a rodentproduces a B cell that expresses an antibody that binds the antigen ofinterest. In some embodiments, an antibody expressed by a B cellincludes a heavy chain having a human heavy chain variable domainencoded by a human heavy chain variable region sequence and/or a lambdalight chain having a human lambda light chain variable domain encoded bya human lambda light chain variable region sequence as described herein.In some embodiments, an antibody expressed by a B cell includes (i) aheavy chain having a human heavy chain variable domain encoded by ahuman heavy chain variable region sequence, (ii) a lambda light chainhaving a human lambda light chain variable domain encoded by a humanlambda light chain variable region sequence as described herein, (iii) akappa light chain having a human kappa light chain variable domainencoded by a human kappa light chain variable region sequence asdescribed herein, or (iv) any combination thereof.

In some embodiments, in response to the step of immunizing, the rodentproduces a population of B cells that expresses antibodies that bind anantigen of interest. In some embodiments, antibodies expressed by apopulation of B cells produced in response to an antigen include a heavychain having a human heavy chain variable domain encoded by a humanheavy chain variable region sequence and/or a lambda light chain havinga human lambda light chain variable domain encoded by a human lambdalight chain variable region sequence as described herein. In someembodiments, antibodies expressed by a population of B cells produced inresponse to an antigen include (i) a heavy chain having a human heavychain variable domain encoded by a human heavy chain variable regionsequence, (ii) a lambda light chain having a human lambda light chainvariable domain encoded by a human lambda light chain variable regionsequence as described herein, (iii) a kappa light chain having a humankappa light chain variable domain encoded by a human kappa light chainvariable region sequence as described herein, or (iv) any combinationthereof.

In some embodiments, in response to the step of immunizing, a rodentproduces a population of B cells that express antibodies that bind toone or more epitopes of antigen of interest, where antibodies expressedby the population of B cells produced in response to an antigen include:(i) a heavy chain having a human heavy chain variable domain encoded bya human heavy chain variable region sequence, (ii) a lambda light chainhaving a human lambda light chain variable domain encoded by a humanlambda light chain variable region sequence as described herein, (iii) akappa light chain having a human kappa light chain variable domainencoded by a human kappa light chain variable region sequence asdescribed herein, or (iv) any combination thereof.

In some embodiments, a human heavy chain variable region sequence, ahuman λ light chain variable region sequence, and/or a human κ lightchain variable region sequence as described herein is somaticallyhypermutated. In some embodiments, at least about 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% ofthe B cells in a population of B cells produced in response to anantigen include a human heavy chain variable region sequence, λ lightchain variable region sequence, and/or κ light chain variable regionsequence that is somatically hypermutated.

In some embodiments, an antibody that binds the antigen of interest isisolated from, recovered from, or identified from a B cell of therodent. In some embodiments, an antibody that binds an antigen ofinterest is isolated from, recovered from, or identified from ahybridoma made with a B cell of the rodent.

In some embodiments, an antigen includes one or more epitopes and anantibody that binds an antigen of interest binds to an epitope of theone or more epitopes.

In some embodiments, a Cλ gene is or includes a rodent Cλ gene. In someembodiments, a rodent Cλ gene has a sequence that is at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, or at least 99%identical to a mouse Cλ1, mouse Cλ2 or a mouse Cλ3 gene. In someembodiments, a rodent Cλ gene is or includes a mouse Cλ1 gene. In someembodiments, a rodent Cλ gene is or includes a rat Cλ gene. In someembodiments, a rat Cλ gene has a sequence that is at least at least 80%,at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%identical to a rat Cλ1, rat Cλ2, rat Cλ3 or a rat Cλ4 gene.

In some embodiments, one or more human Vλ gene segments comprise Vλ5-52,Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39,Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18,Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, Vλ3-1, orany combination thereof. In some embodiments, one or more human Vλ genesegments comprise Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45,Vλ1-44, Vλ7-43, Vλ1-40, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19,Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, andVλ3-1. In some embodiments, one or more human Vλ gene segments compriseVλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40,Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19,Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, andVλ3-1.

In some embodiments, one or more human Jλ gene segments comprise Jλ1,Jλ2, Jλ3, Jλ6, Jλ7 or any combination thereof. In some embodiments, oneor more human Jλ gene segments includes Jλ1, Jλ2, Jλ3, Jλ6, and Jλ7.

In some embodiments, an engineered endogenous immunoglobulin κ lightchain locus includes one or more human Vλ non-coding sequences, whereeach of the one or more human Vλ non-coding sequences is adjacent to theVλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40,Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19,Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3 orVλ3-1 in the engineered endogenous immunoglobulin κ light chain locus,and where each of the one or more human Vλ non-coding sequencesnaturally appear adjacent to a Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46,Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25,Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11,Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3 or Vλ3-1 of an endogenous humanimmunoglobulin λ light chain locus. In some embodiments, an engineeredendogenous immunoglobulin κ light chain locus includes one or more humanJλ non-coding sequences, where each of the one or more human Jλnon-coding sequences is adjacent to the Jλ1, Jλ2, Jλ3, Jλ6 or Jλ7 in theengineered endogenous immunoglobulin κ light chain locus, and where eachof the one or more human Jλ non-coding sequences naturally appearadjacent to a Jλ1, Jλ2, Jλ3, Jλ6 or Jλ7 of an endogenous humanimmunoglobulin λ light chain locus. In some embodiments, an engineeredendogenous immunoglobulin κ light chain locus includes one or more humanJκ non-coding sequences, where each of the one or more human Jκnon-coding sequences is adjacent to the Jλ1, Jλ2, Jλ3, Jλ6 or Jλ7 in theengineered endogenous immunoglobulin κ light chain locus, and where eachof the one or more human Jκ non-coding sequences naturally appearadjacent to a Jκ1, Jκ2, Jκ3, Jκ4, or Jκ5 of an endogenous humanimmunoglobulin κ light chain locus.

In some embodiments, a rodent has a germline genome including anengineered endogenous immunoglobulin heavy chain locus including:

(a) one or more human V_(H) gene segments,

(b) one or more human D_(H) gene segments, and

(c) one or more human J_(H) gene segments,

where the one or more human V_(H) gene segments, the one or more humanD_(H) gene segments, and the one or more human J_(H) gene segments areoperably linked to a rodent immunoglobulin heavy chain constant region.

In some embodiments, one or more human V_(H) gene segments compriseV_(H)3-74, V_(H)3-73, V_(H)3-72, V_(H)2-70, V_(H)1-69, V_(H)3-66,V_(H)3-64, V_(H)4-61, V_(H)4-59, V_(H)1-58, V_(H)3-53, V_(H)5-51,V_(H)3-49, V_(H)3-48, V_(H)1-46, V_(H)1-45, V_(H)3-43, V_(H)4-39,V_(H)4-34, V_(H)3-33, V_(H)4-31, V_(H)3-30, V_(H)4-28, V_(H)2-26,V_(H)1-24, V_(H)3-23, V_(H)3-21, V_(H)3-20, V_(H)1-18, V_(H)3-15,V_(H)3-13, V_(H)3-11, V_(H)3-9, V_(H)1- 8, V_(H)3-7, V_(H)2-5,V_(H)7-4-1, V_(H)4-4, V_(H)1-3, V_(H)1-2, V_(H)6-1 or any combinationthereof. In some embodiments, one or more human V_(H) gene segmentscomprise V_(H)3-74, V_(H)3-73, V_(H)3-72, V_(H)2-70, V_(H)1-69,V_(H)3-66, V_(H)3-64, V_(H)4-61, V_(H)4-59, V_(H)1-58, V_(H)3-53,V_(H)5-51, V_(H)3-49, V_(H)3-48, V_(H)1-46, V_(H)1-45, V_(H)3-43,V_(H)4-39, V_(H)4-34, V_(H)3-33, V_(H)4-31, V_(H)3-30, V_(H)4-28,V_(H)2-26, V_(H)1-24, V_(H)3-23, V_(H)3-21, V_(H)3-20, V_(H)1-18,V_(H)3-15, V_(H)3-13, V_(H)3-11, V_(H)3-9, V_(H)1-8, V_(H)3-7, V_(H)2-5,V_(H)7-4-1, V_(H)4-4, V_(H)1-3, V_(H)1-2, and V_(H)6-1.

In some embodiments, one or more human D_(H) gene segments compriseD_(H)1-1, D_(H)2-2, D_(H)3-3, D_(H)4-4, D_(H)5-5, D_(H)6-6, D_(H)1-7,D_(H)2-8, D_(H)3-9, D_(H)3-10, D_(H)5-12, D_(H)6-13, D_(H)2-15,D_(H)3-16, D_(H)4-17, D_(H)6-19, D_(H)1-20, D_(H)2-21, D_(H)3-22,D_(H)6-25, D_(H)1-26, D_(H)7-27, or any combination thereof. In someembodiments, one or more human D_(H) gene segments comprise D_(H)1-1,D_(H)2-2, D_(H)3-3, D_(H)4-4, D_(H)5-5, D_(H)6-6, D_(H)1-7, D_(H)2-8,D_(H)3-9, D_(H)3-10, D_(H)5-12, D_(H)6-13, D_(H)2-15, D_(H)3-16,D_(H)4-17, D_(H)6-19, D_(H)1-20, D_(H)2-21, D_(H)3-22, D_(H)6-25,D_(H)1-26, and D_(H)7-27.

In some embodiments, one or more human J_(H) gene segments compriseJ_(H)1, J_(H)2, J_(H)3, J_(H)4, J_(H)5, J_(H)6, or any combinationthereof. In some embodiments, one or more human J_(H) gene segmentscomprise J_(H)1, J_(H)2, J_(H)3, J_(H)4, J_(H)5, and J_(H)6.

In some embodiments, an engineered endogenous immunoglobulin heavy chainlocus includes one or more human V_(H) non-coding sequences, where eachof the one or more human V_(H) non-coding sequences is adjacent to theV_(H)3-74, V_(H)3-73, V_(H)3-72, V_(H)2-70, V_(H)1-69, V_(H)3-66,V_(H)3-64, V_(H)4-61, V_(H)4-59, V_(H)1-58, V_(H)3-53, V_(H)5-51,V_(H)3-49, V_(H)3-48, V_(H)1-46, V_(H)1-45, V_(H)3-43, V_(H)4-39,V_(H)4-34, V_(H)3-33, V_(H)4-31, V_(H)3-30, V_(H)4-28, V_(H)2-26,V_(H)1-24, V_(H)3-23, V_(H)3-21, V_(H)3-20, V_(H)1-18, V_(H)3-15,V_(H)3-13, V_(H)3-11, V_(H)3-9, V_(H)1-8, V_(H)3-7, V_(H)2-5,V_(H)7-4-1, V_(H)4-4, V_(H)1-3, V_(H)1-2 or V_(H)6-1 in the engineeredendogenous immunoglobulin heavy chain locus, and where each of the oneor more human V_(H) non-coding sequences naturally appear adjacent to aV_(H)3-74, V_(H)3-73, V_(H)3-72, V_(H)2-70, V_(H)1-69, V_(H)3-66,V_(H)3-64, V_(H)4-61, V_(H)4-59, V_(H)1-58, V_(H)3-53, V_(H)5-51,V_(H)3-49, V_(H)3-48, V_(H)1-46, V_(H)1-45, V_(H)3-43, V_(H)4-39,V_(H)4-34, V_(H)3-33, V_(H)4-31, V_(H)3-30, V_(H)4-28, V_(H)2-26,V_(H)1-24, V_(H)3-23, V_(H)3-21, V_(H)3-20, V_(H)1-18, V_(H)3-15,V_(H)3-13, V_(H)3-11, V_(H)3-9, V_(H)1-8, V_(H)3-7, V_(H)2-5,V_(H)7-4-1, V_(H)4-4, V_(H)1-3, V_(H)1-2 or V_(H)6-1 of an endogenoushuman immunoglobulin heavy chain locus. In some embodiments, anengineered endogenous immunoglobulin heavy chain locus includes one ormore human D_(H) non-coding sequences, where each of the one or morehuman D_(H) non-coding sequences is adjacent to the D_(H)1-1, D_(H)2-2,D_(H)3-3, D_(H)4-4, D_(H)5-5, D_(H)6-6, D_(H)1-7, D_(H)2-8, D_(H)3-9,D_(H)3-10, D_(H)5-12, D_(H)6-13, D_(H)2-15, D_(H)3-16, D_(H)4-17,D_(H)6-19, D_(H)1-20, D_(H)2-21, D_(H)3-22, D_(H)6-25, D_(H)1-26 orD_(H)7-27 in the engineered endogenous immunoglobulin heavy chain locus,and where each of the one or more human D_(H) non-coding sequencesnaturally appear adjacent to a D_(H)1-1, D_(H)2-2, D_(H)3-3, D_(H)4-4,D_(H)5-5, D_(H)6-6, D_(H)1-7, D_(H)2-8, D_(H)3-9, D_(H)3-10, D_(H)5-12,D_(H)6-13, D_(H)2-15, D_(H)3-16, D_(H)4-17, D_(H)6-19, D_(H)1-20,D_(H)2-21, D_(H)3-22, D_(H)6-25, D_(H)1-26 or D_(H)7-27 of an endogenoushuman immunoglobulin heavy chain locus. In some embodiments, anengineered endogenous immunoglobulin heavy chain locus includes one ormore human J_(H) non-coding sequences, where each of the one or morehuman J_(H) non-coding sequences is adjacent to the J_(H)1, J_(H)2,J_(H)3, J_(H)4, J_(H)5 or J_(H)6 in the engineered endogenousimmunoglobulin heavy chain locus, and where each of the one or morehuman J_(H) non-coding sequences naturally appear adjacent to a J_(H)1,J_(H)2, J_(H)3, J_(H)4, J_(H)5 or J_(H)6 of an endogenous humanimmunoglobulin heavy chain locus. In some embodiments, a cell of therodent that is recovered is a B cell. In some embodiments, a cellderived from a cell of the rodent is a hybridoma.

In some embodiments, a nucleotide sequence that encodes a human heavychain variable region sequence, a human lambda light chain variableregion sequence, and/or a human kappa light chain variable regionsequence is obtained from a B cell.

In some embodiments, an engineered endogenous immunoglobulin heavy chainlocus lacks a functional endogenous rodent Adam6 gene. In someembodiments, a germline genome of a rodent includes one or morenucleotide sequences encoding one or more rodent ADAM6 polypeptides,functional orthologs, functional homologs, or functional fragmentsthereof. In some embodiments, one or more rodent ADAM6 polypeptides,functional orthologs, functional homologs, or functional fragmentsthereof are expressed (e.g., in a cell of the male reproductive system,e.g., a testes cell).

In some embodiments, one or more nucleotide sequences encoding one ormore rodent ADAM6 polypeptides, functional orthologs, functionalhomologs, or functional fragments thereof are included on the samechromosome as the engineered endogenous immunoglobulin heavy chainlocus. In some embodiments, one or more nucleotide sequences encodingone or more rodent ADAM6 polypeptides, functional orthologs, functionalhomologs, or functional fragments thereof are included in the engineeredendogenous immunoglobulin heavy chain locus. In some embodiments, one ormore nucleotide sequences encoding one or more rodent ADAM6polypeptides, functional orthologs, functional homologs, or functionalfragments thereof are between a first human V_(H) gene segment and asecond human V_(H) gene segment. In some embodiments, a first humanV_(H) gene segment is V_(H)1-2 and a second human V_(H) gene segment isV_(H)6-1. In some embodiments, one or more nucleotide sequences encodingone or more rodent ADAM6 polypeptides, functional orthologs, functionalhomologs, or functional fragments thereof are in place of a human Adam6pseudogene. In some embodiments, one or more nucleotide sequencesencoding one or more rodent ADAM6 polypeptides, functional orthologs,functional homologs, or functional fragments thereof replace a humanAdam6 pseudogene. In some embodiments, one or more nucleotide sequencesencoding one or more rodent ADAM6 polypeptides, functional orthologs,functional homologs, or functional fragments thereof are between a humanV_(H) gene segment and a human D_(H) gene segment.

In some embodiments, a rodent is a mouse or a rat.

In some embodiments, the present disclosure provides a rodent whosegermline genome includes a homozygous engineered endogenousimmunoglobulin κ light chain locus including:

(i) one or more human Vλ gene segments, where the one or more human Vλgene segments comprise Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45,Vλ1-44, Vλ7-43, Vλ1-40, Vλ5- 39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23,Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10,Vλ3-9, Vλ2-8, Vλ4-3, Vλ3-1, or any combination thereof,

(ii) one or more human Jλ gene segments, where the one or more human Jλgene segments comprise Jλ1, Jλ2, Jλ3, Jλ6, Jλ7, or any combinationthereof, and

(iii) a rodent Cλ gene;

where the one or more human Vλ gene segments, the one or more human Jλgene segments, and the rodent Cλ gene are operably linked to each other,

where the rodent Cλ gene is in place of a rodent Cκ gene of theendogenous immunoglobulin κ light chain locus,

where the engineered endogenous immunoglobulin κ light chain locusincludes:

-   -   (a) one or more human Vλ non-coding sequences, where each of the        one or more human Vλ non-coding sequences is adjacent to the        Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43,        Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22,        Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10,        Vλ3-9, Vλ2-8, Vλ4-3 or Vλ3-1 in the engineered endogenous        immunoglobulin κ light chain locus, and where each of the one or        more human Vλ non-coding sequences naturally appear adjacent to        a Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44,        Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23,        Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11,        Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3 or Vλ3-1 of an endogenous human        immunoglobulin λ light chain locus, and    -   (b) one or more human Jκ non-coding sequences, where each of the        one or more human Jκ non-coding sequences is adjacent to the        Jλ1, Jλ2, Jλ3, Jλ6, or Jλ7 in the engineered endogenous        immunoglobulin κ light chain locus, and where each of the one or        more human Jκ non-coding sequences naturally appear adjacent to        a Jκ 1, Jκ2, Jκ3, Jκ4, or Jκ5 of an endogenous human        immunoglobulin κ light chain locus, and    -   where the immunoglobulin κ light chain locus includes a human κ        light chain non-coding sequence between the one or more human Vλ        gene segments and the one or more human Jλ gene segments that        has a sequence that naturally appears between a human Vκ4-1 gene        segment and a human Jκ1 gene segment in an endogenous human        immunoglobulin κ light chain locus.

In some embodiments, a rodent Cλ gene is a mouse Cλ1 gene.

In some embodiments, an engineered endogenous immunoglobulin κ lightchain locus includes rodent immunoglobulin κ light chain enhancersEκ_(i) and Eκ3′.

In some embodiments, an engineered endogenous immunoglobulin κ lightchain locus includes a deletion of one or more rodent Vκ gene segmentsand/or one or more Jκ gene segments. In some embodiments, an engineeredendogenous immunoglobulin κ light chain locus includes a deletion of allfunctional rodent Vκ and/or Jκ gene segments.

In some embodiments, the present disclosure provides a rodent whosegermline genome includes:

(a) a homozygous endogenous immunoglobulin heavy chain locus includingone or more human V_(H) gene segments, one or more human D_(H) genesegments, and one or more human J_(H) gene segments operably linked toone or more endogenous immunoglobulin heavy chain constant region genessuch that the rodent expresses immunoglobulin heavy chains that eachcomprise a human heavy chain variable domain sequence and a rodent heavychain constant domain sequence,

(b) a first engineered endogenous immunoglobulin κ light chain locusincluding one or more human Vκ gene segments and one or more Jκ genesegments operably linked to an endogenous rodent Cκ region gene suchthat the rodent expresses immunoglobulin light chains that each includesa human κ light chain variable domain sequence and a rodent κ lightchain constant domain sequence, and

(c) a second engineered endogenous immunoglobulin κ light chain locusincluding:

(i) one or more human Vλ gene segments, where the one or more human Vλgene segments comprise Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45,Vλ1-44, Vλ7-43, Vλ1-40, Vλ5- 39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23,Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10,Vλ3-9, Vλ2-8, Vλ4-3, Vλ3-1, or any combination thereof,

(ii) one or more human Jλ gene segments, where the one or more human Jλgene segments comprise Jλ1, Jλ2, Jλ3, Jλ6, Jλ7, or any combinationthereof, and

(iii) a rodent Cλ gene;

where the one or more human Vλ gene segments, the one or more human Jλgene segments, and the rodent Cλ gene are operably linked to each other,

-   -   where the rodent Cλ gene is in place of a rodent Cκ gene of the        endogenous immunoglobulin κ light chain locus,

where the engineered endogenous immunoglobulin κ light chain locusincludes:

-   -   (a) one or more human Vλ non-coding sequences, where each of the        one or more human Vλ non-coding sequences is adjacent to the        Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43,        Vλ1-40, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18,        Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3 or        Vλ3-1 in the engineered endogenous immunoglobulin κ light chain        locus, and where each of the one or more human Vλ non-coding        sequences naturally appear adjacent to a Vλ5-52, Vλ1-51, Vλ9-49,        Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ3-27, Vλ3-25,        Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12,        Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3 or Vλ3-1 of an endogenous        human immunoglobulin λ light chain locus, and    -   (b) one or more human Jκ non-coding sequences, where each of the        one or more human Jκ non-coding sequences is adjacent to the        Jλ1, Jλ2, Jλ3, Jλ6 or Jλ7 in the engineered endogenous        immunoglobulin κ light chain locus, and where each of the one or        more human Jκ non-coding sequences naturally appear adjacent to        a Jκ 1, Jκ2, Jκ3, Jκ4, or Jκ5 of an endogenous human        immunoglobulin κ light chain locus, and    -   where the immunoglobulin κ light chain locus includes a human κ        light chain non-coding sequence between the one or more human Vλ        gene segments and the one or more human Jλ gene segments that        has a sequence that naturally appears between a human Vκ4-1 gene        segment and a human Jκ1 gene segment in an endogenous human        immunoglobulin κ light chain locus;    -   such that the rodent expresses immunoglobulin light chains that        each comprise a human λ light chain variable domain sequence and        a rodent λ light chain constant domain sequence.

In some embodiments, a rodent described herein includes an inactivatedendogenous immunoglobulin λ light chain locus. In some embodiments, arodent described herein is heterozygous for the inactivated endogenousimmunoglobulin λ light chain locus. In some embodiments, a rodentdescribed herein is homozygous for the inactivated endogenousimmunoglobulin λ light chain locus.

In some embodiments, the genome of the rodent further includes a nucleicacid sequence encoding an exogenous terminal deoxynucleotidyltransferase(TdT) operably linked to a transcriptional control element. In someembodiments, the transcriptional control element includes a RAG1transcriptional control element, a RAG2 transcriptional control element,an immunoglobulin heavy chain transcriptional control element, animmunoglobulin κ light chain transcriptional control element, animmunoglobulin λ light chain transcriptional control element, or anycombination thereof. In some embodiments, the nucleic acid sequenceencoding an exogenous TdT is located at an immunoglobulin κ light chainlocus, an immunoglobulin λ light chain locus, an immunoglobulin heavychain locus, a RAG1 locus, or a RAG2 locus. In some embodiments, a TdTis a human TdT. In some embodiments, a TdT is a short isoform of TdT(TdTS).

In some embodiments, a rodent described herein includes a nucleic acidsequence encoding an exogenous terminal deoxynucleotidyltransferase(TdT) operably linked to a transcriptional control element in itsgermline genome and exhibits light chains (e.g., expresses light chainvariable domains including) with at least a 1.2-fold, at least a1.5-fold, at least a 1.75-fold, at least a 2-fold, at least a 3-fold, atleast a 4-fold, or a least a 5-fold increase in junctional diversityover a comparable mouse (e.g., littermate) that does not include anexogenous terminal deoxynucleotidyltransferase (TdT) operably linked toa transcriptional control element in its germline genome. In someembodiments, junctional diversity is measured by number of uniqueCDR3/10,000 reads. In some embodiments, junctional diversity is measuredby number of unique CDR3/10,000 reads.

In some embodiments, a rodent described herein includes a nucleic acidsequence encoding an exogenous terminal deoxynucleotidyltransferase(TdT) operably linked to a transcriptional control element in itsgermline genome and at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 55%, at least 60%, at least65% of light chains (e.g., lambda and/or kappa light chains) produced bythe rodent exhibit non-template additions.

In some embodiments, a rodent described herein is a rat or a mouse.

In some embodiments, the present disclosure provides an antibodyprepared by a method including the steps of:

(a) providing a rodent described herein;

(b) immunizing the rodent with an antigen of interest;

(c) maintaining the rodent under conditions sufficient for the rodent toproduce an immune response to the antigen of interest; and

(d) recovering an antibody that binds the antigen of interest from therodent, or a cell of the rodent, or a cell derived from a cell of therodent,

where the antibody of (d) includes human heavy chain variable and humanλ light chain variable domains.

In some embodiments, the present disclosure provides an antibodyprepared by a method including the steps of:

(a) immunizing a rodent described herein with an antigen of interest;

(b) maintaining the rodent under conditions sufficient for the rodent toproduce an immune response to the antigen of interest; and

(c) recovering an antibody that binds the antigen of interest from therodent, or a cell of the rodent, or a cell derived from a cell of therodent,

where the antibody of (c) includes human heavy chain variable and humanλ light chain variable domains.

In some embodiments, a rodent does not detectably express endogenousimmunoglobulin κ light chain variable domains. In some embodiments, arodent rodent does not detectably express endogenous immunoglobulin λlight chain variable domains.

In some embodiments, a rodent described herein produces a population ofB cells in response to immunization with an antigen that includes one ormore epitopes. In some embodiments, a rodent produces a population of Bcells that express antibodies that bind (e.g., specifically bind) to oneor more epitopes of antigen of interest. In some embodiments, antibodiesexpressed by a population of B cells produced in response to an antigeninclude a heavy chain having a human heavy chain variable domain encodedby a human heavy chain variable region sequence and/or a lambda lightchain having a human lambda light chain variable domain encoded by ahuman lambda light chain variable region sequence as described herein.In some embodiments, antibodies expressed by a population of B cellsproduced in response to an antigen include (i) a heavy chain having ahuman heavy chain variable domain encoded by a human heavy chainvariable region sequence, (ii) a lambda light chain having a humanlambda light chain variable domain encoded by a human lambda light chainvariable region sequence as described herein, (iii) a kappa light chainhaving a human kappa light chain variable domain encoded by a humankappa light chain variable region sequence as described herein, or (iv)any combination thereof.

In some embodiments, a rodent produces a population of B cells thatexpress antibodies that bind to one or more epitopes of antigen ofinterest, where antibodies expressed by the population of B cellsproduced in response to an antigen include: (i) a heavy chain having ahuman heavy chain variable domain encoded by a human heavy chainvariable region sequence, (ii) a lambda light chain having a humanlambda light chain variable domain encoded by a human lambda light chainvariable region sequence as described herein, and/or (iii) a kappa lightchain having a human kappa light chain variable domain encoded by ahuman kappa light chain variable region sequence as described herein.

In some embodiments, a human heavy chain variable region sequence, ahuman λ light chain variable region sequence, and/or a human κ lightchain variable region sequence as described herein is somaticallyhypermutated. In some embodiments, at least about 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% ofthe B cells in a population of B cells produced in response to anantigen include a human heavy chain variable region sequence, λ lightchain variable region sequence, and/or κ light chain variable regionsequence that is somatically hypermutated.

In some embodiments, the present disclosure provides a method of makingan antibody, including:

(i) expressing a first nucleotide sequence that encodes animmunoglobulin heavy chain in a host cell, where the first nucleotidesequence includes a human heavy chain variable region sequence;

(ii) expressing a second nucleotide sequence that encodes animmunoglobulin λ light chain in a host cell, where the second nucleotidesequence includes a human λ light chain variable region sequence thatwas identified (e.g., expressed and/or isolated) from a rodent whosegermline genome includes:

-   -   an engineered endogenous immunoglobulin κ light chain locus        including:        -   (a) one or more human Vλ gene segment,        -   (b) one or more human Jλ gene segment, and        -   (c) one or more Cλ genes,    -   where the one or more human Vλ gene segment and the one or more        human Jλ gene segment are operably linked to the one or more Cλ        genes, and    -   where the rodent lacks a rodent Cκ gene at the engineered        endogenous immunoglobulin κ locus;

(iii) culturing the host cell so that immunoglobulin light chains andimmunoglobulin heavy chains are expressed and form an antibody; and

(iv) obtaining the antibody from the host cell and/or host cell culture.

In some embodiments, a first nucleotide sequence includes a human heavychain constant region. In some embodiments, an antibody is a fully humanantibody.

In some embodiments, a second nucleotide includes a human λ light chainconstant region sequence.

In some embodiments, an antibody is a reverse chimeric antibody. In someembodiments, a first nucleotide sequence includes a rodent heavy chainconstant region. In some embodiments, a second nucleotide sequenceincludes a rodent λ light chain constant region sequence.

In some embodiments, the present disclosure provides a rodent, whosegermline genome includes:

(a) a first engineered endogenous immunoglobulin κ light chain locuscomprising:

-   -   (i) one or more human Vλ gene segments,    -   (ii) one or more human Jλ gene segments, and    -   (iii) a Cλ gene,    -   where the one or more human Vλ gene segments and the one or more        human Jλ gene segments are operably linked to the Cλ gene, and

where the rodent lacks a rodent Cκ gene at the first engineeredendogenous immunoglobulin κ locus; and

(b) a second engineered endogenous immunoglobulin κ light chain locusfurther includes:

-   -   (i) one or more human Vκ gene segments, and    -   (ii) one or more human Jκ gene segments,    -   where the one or more human Vκ gene segments and the one or more        human Jκ gene segments are operably linked to a Cκ gene.

In some embodiments, a Cκ gene is an endogenous rodent Cκ gene.

In some embodiments, the genome of the rodent further includes a nucleicacid sequence encoding an exogenous terminal deoxynucleotidyltransferase(TdT) operably linked to a transcriptional control element. In someembodiments, the transcriptional control element includes a RAG1transcriptional control element, a RAG2 transcriptional control element,an immunoglobulin heavy chain transcriptional control element, animmunoglobulin κ light chain transcriptional control element, animmunoglobulin λ light chain transcriptional control element, or anycombination thereof. In some embodiments, the nucleic acid sequenceencoding an exogenous TdT is located at an immunoglobulin κ light chainlocus, an immunoglobulin λ light chain locus, an immunoglobulin heavychain locus, a RAG1 locus, or a RAG2 locus. In some embodiments, a TdTis a human TdT. In some embodiments, a TdT is a short isoform of TdT(TdTS).

In some embodiments, the genome of the rodent further includes a nucleicacid sequence encoding an exogenous terminal deoxynucleotidyltransferase(TdT) operably linked to a transcriptional control element. In someembodiments, the transcriptional control element includes a RAG1transcriptional control element, a RAG2 transcriptional control element,an immunoglobulin heavy chain transcriptional control element, animmunoglobulin κ light chain transcriptional control element, animmunoglobulin λ light chain transcriptional control element, or anycombination thereof. In some embodiments, the nucleic acid sequenceencoding an exogenous TdT is located at an immunoglobulin κ light chainlocus, an immunoglobulin λ light chain locus, an immunoglobulin heavychain locus, a RAG1 locus, or a RAG2 locus. In some embodiments, a TdTis a human TdT. In some embodiments, a TdT is a short isoform of TdT(TdTS).

In some embodiments, a rodent described herein includes a nucleic acidsequence encoding an exogenous terminal deoxynucleotidyltransferase(TdT) operably linked to a transcriptional control element in itsgermline genome and exhibits light chains (e.g., expresses light chainvariable domains including) with at least a 1.2-fold, at least a1.5-fold, at least a 1.75-fold, at least a 2-fold, at least a 3-fold, atleast a 4-fold, or a least a 5-fold increase in junctional diversityover a comparable mouse (e.g., littermate) that does not include anexogenous terminal deoxynucleotidyltransferase (TdT) operably linked toa transcriptional control element in its germline genome. In someembodiments, junctional diversity is measured by number of uniqueCDR3/10,000 reads. In some embodiments, junctional diversity is measuredby number of unique CDR3/10,000 reads.

In some embodiments, a rodent described herein includes a nucleic acidsequence encoding an exogenous terminal deoxynucleotidyltransferase(TdT) operably linked to a transcriptional control element in itsgermline genome and at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 55%, at least 60%, at least65% of light chains (e.g., lambda and/or kappa light chains) produced bythe rodent exhibit non-template additions.

In various embodiments, a non-human animal, non-human cell or non-humantissue as described herein is a rodent, rodent cell or rodent tissue; insome embodiments, a mouse, mouse cell or mouse tissue; in someembodiments, a rat, rat cell or rat tissue. In some embodiments, amouse, mouse cell or mouse tissue as described herein comprises agenetic background that includes a 129 strain, a BALB/c strain, aC57BL/6 strain, a mixed 129xC57BL/6 strain, or combinations thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The Drawings included herein, which are composed of the followingFigures, is for illustration purposes only and not for limitation.

FIGS. 1A and 1B show illustrations of an exemplary embodiment, not toscale, of a strategy for constructing a targeting vector (described inExample 1.1) used in generating an embodiment of the rodent according tothe present disclosure.

FIG. 2A shows an illustration of an exemplary embodiment, not to scale,of the insertion of a targeting vector (described in Example 1.1) intoan engineered Igκ light chain locus of a rodent embryonic stem (ES) cellclone, which ES cell clone was used in generating an embodimentaccording to the present disclosure.

FIG. 2B shows an illustration of an exemplary embodiment, not to scale,of recombinase-mediated removal of selection cassette(s) in anengineered Igκ light chain locus resulting from the insertion of atargeting vector (described in Example 1.1) used in generating anembodiment of the rodent according to the present disclosure.

FIG. 3 shows an illustration of an exemplary embodiment, not to scale,of a strategy for constructing a targeting vector (described in Example1.2) used in generating an embodiment of the rodent according to thepresent disclosure.

FIG. 4A shows an illustration, not to scale, of the insertion of atargeting vector (described in Example 1.2) into an engineered Igκ lightchain locus of a rodent embryonic stem (ES) cell clone, which ES cellclone was used in generating an embodiment of the rodent according tothe present disclosure.

FIG. 4B shows an illustration of an exemplary embodiment, not to scale,of recombinase-mediated removal of selection cassette(s) in anengineered Igκ light chain locus resulting from the insertion of atargeting vector (described in Example 1.2) used in generating anembodiment of the rodent according to the present disclosure.

FIG. 5 shows results derived from a representative embodiment accordingto the present disclosure, showing single cell-gated splenocytesharvested from wild-type (WT) and 6558 HO (LiK, homozygous) mice, thetop row illustrating expression of CD19 (y-axis) and CD3 (x-axis), andthe bottom row illustrating CD19⁺-gated splenocytes expressingimmunoglobulin D (IgD, y-axis) and immunoglobulin M (IgM, x-axis).

FIG. 6 shows results derived from a representative embodiment accordingto the present disclosure, including representative single cell-gatedbone marrow harvested from wild-type (WT) and 6558HO (LiK, homozygous)mice, the top row illustrating expression of CD19 (y-axis) and CD3(x-axis), and the bottom row illustrating expression of immunoglobulin M(IgD, y-axis) and B220 (x-axis).

FIG. 7 shows results derived from a representative embodiment accordingto the present disclosure, including representative CD19⁺-gatedsplenocytes harvested from wild-type (WT) and 6558HO (LiK, homozygous)mice illustrating expression of immunoglobulin light chains containingmouse Igλ (y-axis) or mouse Igκ (x-axis) constant regions.

FIG. 8 shows results derived from a representative embodiment accordingto the present disclosure, including representative single cell-gatedsplenocytes harvested from various indicated humanized mice illustratingexpression of CD19 (y-axis) and CD3 (x-axis). HOH/LiK/λ^(−/−) mice-micehomozygous for humanized immunoglobulin heavy chain (see, e.g., U.S.Pat. Nos. 8,642,835 and 8,697,940), homozygous for LiK locus andhomozygous for an inactivated endogenous immunoglobulin λ light chainlocus; HOH/KoK/LiK/λ^(−/−) mice-mice homozygous for humanizedimmunoglobulin heavy chain (see, e.g., U.S. Pat. Nos. 8,642,835 and8,697,940), hemizygous for one kappa locus comprising LiK locus and asecond kappa locus comprising humanized immunoglobulin kappa light chainlocus, and homozygous for an inactivated endogenous immunoglobulin λlight chain locus; HOH/KoK mice-control mice homozygous for humanizedimmunoglobulin heavy chain and homozygous for humanized immunoglobulinkappa light chain.

FIG. 9 shows results derived from a representative embodiment accordingto the present disclosure, including representative CD19⁺-gatedsplenocytes harvested from various indicated humanized mice illustratingexpression of immunoglobulin light chains containing mouse Igλ (y-axis)or mouse Igκ (x-axis) constant regions.

FIG. 10 shows results derived from a representative embodiment accordingto the present disclosure, including representative single cell-gatedbone marrow harvested from various indicated humanized mice illustratingexpression of immunoglobulin M (IgD, y-axis) and B220 (x-axis).

FIG. 11 shows results derived from a representative embodiment accordingto the present disclosure, including representative single cell-gatedbone marrow harvested from various indicated humanized mice illustratingexpression of immunoglobulin light chains containing mouse Igλ (y-axis)or mouse Igκ (x-axis) constant regions in immature (top row) and mature(bottom row) B cells.

FIG. 12 shows a schematic illustration of an exemplary embodiment,according to the present disclosure, not to scale, of an engineeredimmunoglobulin κ light chain locus as described herein and therearrangement of the locus to form an mRNA molecule.

FIG. 13 shows results derived from a representative embodiment accordingto the present disclosure, including representative protein immunoblots(Western blots) of SDS-PAGE using serum isolated from wild-type (WT) and6558 homozygous (LiK HO) mice as described in Example 3.3.

FIG. 14 shows results of testing an embodiment according to the presentdisclosure, showing representative single cell-gated splenocytesharvested from humanized mice illustrating expression of CD19 (y-axis)and CD3 (x-axis). HOH/LiK/λ^(−/−)/TdT mice-mice homozygous for humanizedimmunoglobulin heavy chain (see, e.g., U.S. Pat. Nos. 8,642,835 and8,697,940), homozygous for LiK locus and homozygous for an inactivatedendogenous immunoglobulin λ light chain locus that include a nucleicacid sequence encoding an exogenous terminal deoxynucleotidyltransferase(TdT); and HOH/KoK/LiK/λ^(−/−)/TdT mice-mice homozygous for humanizedimmunoglobulin heavy chain (see, e.g., U.S. Pat. Nos. 8,642,835 and8,697,940), hemizygous for one kappa locus comprising an LiK locus and asecond kappa locus comprising humanized immunoglobulin kappa light chainlocus, and homozygous for an inactivated endogenous immunoglobulin λlight chain locus that include a nucleic acid sequence encoding anexogenous terminal deoxynucleotidyltransferase (TdT).

FIG. 15 shows results of testing an embodiment according to the presentdisclosure, showing representative CD19⁺-gated splenocytes harvestedfrom various indicated humanized mice illustrating expression ofimmunoglobulin light chains containing mouse Igλ (y-axis) or mouse Igκ(x-axis) constant regions.

FIG. 16 shows results of testing an embodiment according to the presentdisclosure, showing representative single cell-gated bone marrowharvested from various indicated humanized mice illustrating expressionof immunoglobulin M (IgM, y-axis) and B220 (x-axis).

FIG. 17 shows results of testing an embodiment according to the presentdisclosure, showing representative single cell-gated bone marrowharvested from various indicated humanized mice illustrating expressionof immunoglobulin light chains containing mouse Igλ (y-axis) or mouseIgκ (x-axis) constant regions in immature (top row) and mature (bottomrow) B cells.

FIG. 18 shows results of testing an embodiment according to the presentdisclosure, showing a graph comparing immune responses in LiK/VI-3,LiK/VI-3/TdT and VI-3/TdT mice strains following immunization with aprotein immunogen.

FIG. 19 shows results of testing an embodiment according to the presentdisclosure, showing a graph comparing immune responses against His tagin LiK/VI-3, LiK/VI-3/TdT and VI-3/TdT mice strains followingimmunization with an irrelevant protein antigen fused to a HIS tag.

FIG. 20 shows an illustration, not to scale, of a portion of anendogenous human immunoglobulin λ light chain locus. FIG. 20 includes afirst arrow pointing to a representation of a first exemplary endogenoushuman Vλ non-coding sequence in the endogenous human immunoglobulin λlight chain locus. As illustrated, the first exemplary endogenous humanVλ non-coding sequence (represented by a line) in the endogenous humanimmunoglobulin λ light chain locus naturally appears adjacent to a humanVλ3-12 gene segment (represented by a dark grey square) and a humanVλ2-11 gene segment (represented by a dark grey square) in theendogenous human immunoglobulin Igλ light chain locus. FIG. 20 alsoincludes a second arrow pointing to a representation of a secondexemplary endogenous human Vλ non-coding sequence in the endogenoushuman immunoglobulin λ light chain locus. As illustrated, the secondexemplary endogenous human Vλ non-coding sequence (represented by aline) in the endogenous human immunoglobulin λ light chain locusnaturally appears adjacent to a human Vλ2-11 gene segment (representedby a dark grey square) and a human Vλ3-10 gene segment (represented by adark grey square) in the endogenous human immunoglobulin λ light chainlocus.

FIG. 21 shows an illustration, not to scale, of a portion of anendogenous human immunoglobulin κ light chain locus. FIG. 21 includes afirst arrow pointing to a representation of a first exemplary endogenoushuman Jκ non-coding sequence in the endogenous human immunoglobulin κlight chain locus. As illustrated, the first exemplary endogenous humanJκ non-coding sequence (represented by a line) in the endogenous humanimmunoglobulin κ light chain locus naturally appears adjacent to a humanJκ1 gene segment (represented by a dark grey square) and a human Jκ2gene segment (represented by a dark grey square) in the endogenous humanimmunoglobulin κ light chain locus. FIG. 21 also includes a second arrowpointing to a representation of a second exemplary endogenous human Jκnon-coding sequence in the endogenous human immunoglobulin κ light chainlocus. As illustrated, the second exemplary endogenous human Jκnon-coding sequence (represented by a line) in the endogenous humanimmunoglobulin κ light chain locus naturally appears adjacent to a humanJκ2 gene segment (represented by a dark grey square) and a human Jκ3gene segment (represented by a dark grey square) in the endogenous humanimmunoglobulin κ light chain locus.

BRIEF DESCRIPTION OF SELECTED SEQUENCES IN THE SEQUENCE LISTING

The following are representative nucleic acid and amino acid sequence ofvarious immunoglobulin constant regions of the mouse, rat, or humanlambda genes. Nucleic acid and amino acid sequences of immunoglobulingenes and polypeptides are available from the

Mouse Cλ1 DNA (SEQ ID NO: 1):GCCAGCCCAAGTCTTCGCCATCAGTCACCCTGTTTCCACCTTCCTCTGAAGAGCTCGAGACTAACAAGGCCACACTGGTGTGTACGATCACTGATTTCTACCCAGGTGTGGTGACAGTGGACTGGAAGGTAGATGGTACCCCTGTCACTCAGGGTATGGAGACAACCCAGCCTTCCAAACAGAGCAACAACAAGTACATGGCTAGCAGCTACCTGACCCTGACAGCAAGAGCATGGGAAAGGCATAGCAGTTACAGCTGCCAGGTCACTCATGAAGGTCACACTGTGGAGAAGAGTTTGTCCCGTGCTGACTGTTCCMouse Cλ1 amino acid (SEQ ID NO: 2):GQPKSSPSVTLFPPSSEELETNKATLVCTITDFYPGVVTVDWKVDGTPVTQGMETTQPSKQSNNKYMASSYLTLTARAWERHSSYSCQVT HEGHTVEKSL SRADCS Mouse Cλ2 DNA (SEQ ID NO: 3):GTCAGCCCAAGTCCACTCCCACTCTCACCGTGTTTCCACCTTCCTCTGAGGAGCTCAAGGAAAACAAAGCCACACTGGTGTGTCTGATTTCCAACTTTTCCCCGAGTGGTGTGACAGTGGCCTGGAAGGCAAATGGTACACCTATCACCCAGGGTGTGGACACTTCAAATCCCACCAAAGAGGGCAACAAGTTCATGGCCAGCAGCTTCCTACATTTGACATCGGACCAGTGGAGATCTCACAACAGTTTTACCTGTCAAGTTACACATGAAGGGGACACTGTGGAGAAGAGTCTGTCTCCTGCAGAATGTCTCMouse Cλ2 amino acid (SEQ ID NO: 4):GQPKSTPTLTVFPPSSEELKENKATLVCLISNFSPSGVTVAWKANGTPITQGVDTSNPTKEGNKFMASSFLHLTSDQWRSHNSFTCQVTHEGDTVEKSLSPAECL Mouse Cλ3 DNA (SEQ ID NO: 5):GTCAGCCCAAGTCCACTCCCACACTCACCATGTTTCCACCTTCCCCTGAGGAGCTCCAGGAAAACAAAGCCACACTCGTGTGTCTGATTTCCAATTTTTCCCCAAGTGGTGTGACAGTGGCCTGGAAGGCAAATGGTACACCTATCACCCAGGGTGTGGACACTTCAAATCCCACCAAAGAGGACAACAAGTACATGGCCAGCAGCTTCTTACATTTGACATCGGACCAGTGGAGATCTCACAACAGTTTTACCTGCCAAGTTACACATGAAGGGGACACTGTGGAGAAGAGTCTGTCTCCTGCAGAATGTCTCMouse Cλ3 amino acid (SEQ ID NO: 6):GQPKSTPTLTMFPPSPEELQENKATLVCLISNFSPSGVTVAWKANGTPITQGVDTSNPTKEDNKYMASSFLHLTSDQWRSHNSFTCQVTHEGDTVEKSLSPAECL Rat Cλ1 DNA (SEQ ID NO: 7):GTCAGCCCAAGTCCACTCCCACACTCACAGTATTTCCACCTTCAACTGAGGAGCTCCAGGGAAACAAAGCCACACTGGTGTGTCTGATTTCTGATTTCTACCCGAGTGATGTGGAAGTGGCCTGGAAGGCAAATGGTGCACCTATCTCCCAGGGTGTGGACACTGCAAATCCCACCAAACAGGGCAACAAATACATCGCCAGCAGCTTCTTACGTTTGACAGCAGAACAGTGGAGATCTCGCAACAGTTTTACCTGCCAAGTTACACATGAAGGGAACACTGTGGAGAAGAGTCTGTCTCCTGCAGAATGTGTCRat Cλ1 amino acid (SEQ ID NO: 8):GQPKSTPTLTVFPPSTEELQGNKATLVCLISDFYPSDVEVAWKANGAPISQGVDTANPTKQGNKYIASSFLRLTAEQWRSRNSFTCQVTHEGNTVEKSLSPAECV Rat Cλ2 DNA (SEQ ID NO: 9):ACCAACCCAAGGCTACGCCCTCAGTCACCCTGTTCCCACCTTCCTCTGAAGAGCTCAAGACTGACAAGGCTACACTGGTGTGTATGGTGACAGATTTCTACCCTGGTGTTATGACAGTGGTCTGGAAGGCAGATGGTACCCCTATCACTCAGGGTGTGGAGACTACCCAGCCTTTCAAACAGAACAACAAGTACATGGCTACCAGCTACCTGCTTTTGACAGCAAAAGCATGGGAGACTCATAGCAATTACAGCTGCCAGGTCACTCACGAAGAGAACACTGTGGAGAAGAGTTTGTCCCGTGCTGAGTGTTCCRat Cλ2 amino acid (SEQ ID NO: 10):DQPKATPSVTLFPPSSEELKTDKATLVCMVTDFYPGVMTVVWKADGTPITQGVETTQPFKQNNKYMATSYLLLTAKAWETHSNYSCQVTHEENTVEKSLSRAECS Rat Cλ3 DNA (SEQ ID NO: 11):GTCAGCCCAAGTCCACTCCCACACTCACAGTATTTCCACCTTCAACTGAGGAGCTCCAGGGAAACAAAGCCACACTGGTGTGTCTGATTTCTGATTTCTACCCGAGTGATGTGGAAGTGGCCTGGAAGGCAAATGGTGCACCTATCTCCCAGGGTGTGGACACTGCAAATCCCACCAAACAGGGCAACAAATACATCGCCAGCAGCTTCTTACGTTTGACAGCAGAACAGTGGAGATCTCGCAACAGTTTTACCTGCCAAGTTACACATGAAGGGAACACTGTGGAAAAGAGTCTGTCTCCTGCAGAGTGTGTCRat Cλ3 amino acid (SEQ ID NO: 12):GQPKSTPTLTVFPPSTEELQGNKATLVCLISDFYPSDVEVAWKANGAPISQGVDTANPTKQGNKYIASSFLRLTAEQWRSRNSFTCQVTHEGNTVEKSLSPAECV Rat Cλ4 DNA (SEQ ID NO: 13):ACCAACCCAAGGCTACGCCCTCAGTCACCCTGTTCCCACCTTCCTCTGAAGAGCTCAAGACTGACAAGGCTACACTGGTGTGTATGGTGACAGATTTCTACCCTGGTGTTATGACAGTGGTCTGGAAGGCAGATGGTACCCCTATCACTCAGGGTGTGGAGACTACCCAGCCTTTCAAACAGAACAACAAGTACATGGCTACCAGCTACCTGCTTTTGACAGCAAAAGCATGGGAGACTCATAGCAATTACAGCTGCCAGGTCACTCACGAAGAGAACACTGTGGAGAAGAGTTTGTCCCGTGCTGAGTGTTCCRat Cλ4 amino acid (SEQ ID NO: 14):DQPKATPSVTLFPPSSEELKTDKATLVCMVTDFYPGVMTVVWKADGTPITQGVETTQPFKQNNKYMATSYLLLTAKAWETHSNYSCQVTHEENTVEKSLSRAECS Human Cλ1 DNA (SEQ ID NO: 15):CCCAAGGCCAACCCCACGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTCCAAGCCAACAAGGCCACACTAGTGTGTCTGATCAGTGACTTCTACCCGGGAGCTGTGACAGTGGCTTGGAAGGCAGATGGCAGCCCCGTCAAGGCGGGAGTGGAGACGACCAAACCCTCCAAACAGAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCCGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCATAGHuman Cλ1 amino acid (SEQ ID NO: 16):PKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS Human Cλ2 DNA (SEQ ID NO: 17):GTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCTTGGAAAGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCAHuman Cλ2 amino acid (SEQ ID NO: 18):QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS Human Cλ3 DNA (SEQ ID NO: 19):CCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCACCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTTGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGGGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAAAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTTGCCCCTACGGAATGTTCATAGHuman Cλ3 amino acid (SEQ ID NO: 20):PKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAPTECS Human Cλ6 DNA (SEQ ID NO: 21):GGTCAGCCCAAGGCTGCCCCATCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGCCTGATCAGTGACTTCTACCCGGGAGCTGTGAAAGTGGCCTGGAAGGCAGATGGCAGCCCCGTCAACACGGGAGTGGAGACCACCACACCCTCCAAACAGAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTGCAGAATGTTCATAG Human Cλ6 amino acid (SEQ ID NO: 22):QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVKVAWKADGSPVNTGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPAECS Human Cλ7 DNA (SEQ ID NO: 23):GTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCACCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCGTAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATGGCAGCCCCGTCAAGGTGGGAGTGGAGACCACCAAACCCTCCAAACAAAGCAACAACAAGTATGCGGCCAGCAGCTACCTGAGCCTGACGCCCGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCGGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTGCAGAATGCTCTHuman Cλ7 amino acid (SEQ ID NO: 24):QPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADGSPVKVGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCRVTHEGSTVEKTVAPAECS

Definitions

The scope of the present invention is defined by the claims appendedhereto and is not limited by certain embodiments described herein. Thoseskilled in the art, reading the present specification, will be aware ofvarious modifications that may be equivalent to such describedembodiments, or otherwise within the scope of the claims. In general,terms used herein are in accordance with their understood meaning in theart, unless clearly indicated otherwise. Explicit definitions of certainterms are provided below; meanings of these and other terms inparticular instances throughout this specification will be clear tothose skilled in the art from context. Additional definitions for thefollowing and other terms are set forth throughout the specification.Patent and non-patent literature references cited within thisspecification, or relevant portions thereof, are incorporated herein byreference in their entireties.

Use of ordinal terms such as “first,” “second,” “third,” etc., in theclaims to modify a claim element does not by itself connote anypriority, precedence, or order of one claim element over another or thetemporal order in which acts of a method are performed, but are usedmerely as labels to distinguish one claim element having a certain namefrom another element having a same name (but for use of the ordinalterm) to distinguish the claim elements.

As used in this application, the terms “about” and “approximately” areused as equivalents. Any numerals used in this application with orwithout about or approximately are meant to cover any normalfluctuations appreciated by one of ordinary skill in the relevant art.

The articles “a” and “an” in the specification and in the claims, unlessclearly indicated to the contrary, should be understood to include theplural referents. Claims or descriptions that include “or” between oneor more members of a group are considered satisfied if one, more thanone, or all of the group members are present in, employed in, orotherwise relevant to a given product or process unless indicated to thecontrary or otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention also includes embodiments in which more than one, or theentire group members are present in, employed in, or otherwise relevantto a given product or process. Furthermore, it is to be understood thatthe invention encompasses all variations, combinations, and permutationsin which one or more limitations, elements, clauses, descriptive terms,etc., from one or more of the listed claims is introduced into anotherclaim dependent on the same base claim (or, as relevant, any otherclaim) unless otherwise indicated or unless it would be evident to oneof ordinary skill in the art that a contradiction or inconsistency wouldarise. Where elements are presented as lists, (e.g., in Markush group orsimilar format) it is to be understood that each subgroup of theelements is also disclosed, and any element(s) can be removed from thegroup. It should be understood that, in general, where the invention, oraspects of the invention, is/are referred to as comprising particularelements, features, etc., certain embodiments of the invention oraspects of the invention consist, or consist essentially of, suchelements, features, etc. For purposes of simplicity those embodimentshave not in every case been specifically set forth in so many wordsherein. It should also be understood that any embodiment or aspect ofthe invention can be explicitly excluded from the claims, regardless ofwhether the specific exclusion is recited in the specification.

Administration: as used herein, includes the administration of acomposition to a subject or system (e.g., to a cell, organ, tissue,organism, or relevant component or set of components thereof). Theskilled artisan will appreciate that route of administration may varydepending, for example, on the subject or system to which thecomposition is being administered, the nature of the composition, thepurpose of the administration, etc. For example, in certain embodiments,administration to an animal subject (e.g., to a human or a rodent) maybe bronchial (including by bronchial instillation), buccal, enteral,interdermal, intra-arterial, intradermal, intragastric, intramedullary,intramuscular, intranasal, intraperitoneal, intrathecal, intravenous,intraventricular, mucosal, nasal, oral, rectal, subcutaneous,sublingual, topical, tracheal (including by intratracheal instillation),transdermal, vaginal and/or vitreal. In some embodiments, administrationmay involve intermittent dosing. In some embodiments, administration mayinvolve continuous dosing (e.g., perfusion) for at least a selectedperiod of time.

Amelioration: as used herein, includes the prevention, reduction orpalliation of a state, or improvement of the state of a subject.Amelioration includes but does not require complete recovery or completeprevention of a disease, disorder or condition.

Approximately: as applied to one or more values of interest, includes toa value that is similar to a stated reference value. In certainembodiments, the term “approximately” or “about” refers to a range ofvalues that fall within ±10% (greater than or less than) of the statedreference value unless otherwise stated or otherwise evident from thecontext (except where such number would exceed 100% of a possiblevalue).

Biologically active: as used herein, refers to a characteristic of anyagent that has activity in a biological system, in vitro or in vivo(e.g., in an organism). For instance, an agent that, when present in anorganism, has a biological effect within that organism is considered tobe biologically active. In particular embodiments, where a protein orpolypeptide is biologically active, a portion of that protein orpolypeptide that shares at least one biological activity of the proteinor polypeptide is typically referred to as a “biologically active”portion.

Comparable: as used herein, refers to two or more agents, entities,situations, sets of conditions, etc. that may not be identical to oneanother but that are sufficiently similar to permit comparison therebetween so that conclusions may reasonably be drawn based on differencesor similarities observed. Persons of ordinary skill in the art willunderstand, in context, what degree of identity is required in any givencircumstance for two or more such agents, entities, situations, sets ofconditions, etc. to be considered comparable.

Conservative: as used herein, refers to instances when describing aconservative amino acid substitution, including a substitution of anamino acid residue by another amino acid residue having a side chain Rgroup with similar chemical properties (e.g., charge or hydrophobicity).In general, a conservative amino acid substitution will notsubstantially change the functional properties of interest of a protein,for example, the ability of a receptor to bind to a ligand. Examples ofgroups of amino acids that have side chains with similar chemicalproperties include: aliphatic side chains such as glycine (Gly, G),alanine (Ala, A), valine (Val, V), leucine (Leu, L), and isoleucine(Ile, I); aliphatic-hydroxyl side chains such as serine (Ser, S) andthreonine (Thr, T); amide-containing side chains such as asparagine(Asn, N) and glutamine (Gln, Q); aromatic side chains such asphenylalanine (Phe, F), tyrosine (Tyr, Y), and tryptophan (Trp, W);basic side chains such as lysine (Lys, K), arginine (Arg, R), andhistidine (His, H); acidic side chains such as aspartic acid (Asp, D)and glutamic acid (Glu, E); and sulfur-containing side chains such ascysteine (Cys, C) and methionine (Met, M). Conservative amino acidssubstitution groups include, for example, valine/leucine/isoleucine(Val/Leu/Ile, V/L/I), phenylalanine/tyrosine (Phe/Tyr, F/Y),lysine/arginine (Lys/Arg, K/R), alanine/valine (Ala/Val, A/V),glutamate/aspartate (Glu/Asp, E/D), and asparagine/glutamine (Asn/Gln,N/Q). In some embodiments, a conservative amino acid substitution can bea substitution of any native residue in a protein with alanine, as usedin, for example, alanine scanning mutagenesis. In some embodiments, aconservative substitution is made that has a positive value in thePAM250 log-likelihood matrix disclosed in Gonnet, G. H. et al., 1992,Science 256:1443-1445, which is incorporated herein by reference in itsentirety. In some embodiments, a substitution is a moderatelyconservative substitution wherein the substitution has a nonnegativevalue in the PAM250 log-likelihood matrix.

Control: as used herein, refers to the art-understood meaning of a“control” being a standard against which results are compared.Typically, controls are used to augment integrity in experiments byisolating variables in order to make a conclusion about such variables.In some embodiments, a control is a reaction or assay that is performedsimultaneously with a test reaction or assay to provide a comparator. A“control” also includes a “control animal.” A “control animal” may havea modification as described herein, a modification that is different asdescribed herein, or no modification (i.e., a wild-type animal). In oneexperiment, a “test” (i.e., a variable being tested) is applied. In asecond experiment, the “control,” the variable being tested is notapplied. In some embodiments, a control is a historical control (i.e.,of a test or assay performed previously, or an amount or result that ispreviously known). In some embodiments, a control is or comprises aprinted or otherwise saved record. A control may be a positive controlor a negative control.

Disruption: as used herein, refers to the result of a homologousrecombination event with a DNA molecule (e.g., with an endogenoushomologous sequence such as a gene or gene locus). In some embodiments,a disruption may achieve or represent an insertion, deletion,substitution, replacement, missense mutation, or a frame-shift of a DNAsequence(s), or any combination thereof. Insertions may include theinsertion of entire genes or gene fragments, e.g., exons, which may beof an origin other than the endogenous sequence (e.g., a heterologoussequence). In some embodiments, a disruption may increase expressionand/or activity of a gene or gene product (e.g., of a polypeptideencoded by a gene). In some embodiments, a disruption may decreaseexpression and/or activity of a gene or gene product. In someembodiments, a disruption may alter sequence of a gene or an encodedgene product (e.g., an encoded polypeptide). In some embodiments, adisruption may truncate or fragment a gene or an encoded gene product(e.g., an encoded polypeptide). In some embodiments, a disruption mayextend a gene or an encoded gene product. In some such embodiments, adisruption may achieve assembly of a fusion polypeptide. In someembodiments, a disruption may affect level, but not activity, of a geneor gene product. In some embodiments, a disruption may affect activity,but not level, of a gene or gene product. In some embodiments, adisruption may have no significant effect on level of a gene or geneproduct. In some embodiments, a disruption may have no significanteffect on activity of a gene or gene product. In some embodiments, adisruption may have no significant effect on either level or activity ofa gene or gene product.

Determining, measuring, evaluating, assessing, assaying and analyzing:are used interchangeably herein to refer to any form of measurement, andinclude determining if an element is present or not. These terms includeboth quantitative and/or qualitative determinations. Assaying may berelative or absolute. “Assaying for the presence of” can be determiningthe amount of something present and/or determining whether or not it ispresent or absent.

Endogenous promoter: as used herein, refers to a promoter that isnaturally associated, e.g., in a wild-type organism, with an endogenousgene.

Engineered: as used herein refers, in general, to the aspect of havingbeen manipulated by the hand of man. For example, in some embodiments, apolynucleotide may be considered to be “engineered” when two or moresequences that are not linked together in that order in nature aremanipulated by the hand of man to be directly linked to one another inthe engineered polynucleotide. In some embodiments, an engineeredpolynucleotide may comprise a regulatory sequence that is found innature in operative association with a first coding sequence but not inoperative association with a second coding sequence, is linked by thehand of man so that it is operatively associated with the second codingsequence. Alternatively, or additionally, in some embodiments, first andsecond nucleic acid sequences that each encode polypeptide elements ordomains that in nature are not linked to one another may be linked toone another in a single engineered polynucleotide. Comparably, in someembodiments, a cell or organism may be considered to be “engineered” ifit has been manipulated so that its genetic information is altered(e.g., new genetic material not previously present has been introduced,or previously present genetic material has been altered or removed). Asis common practice and is understood by persons of skill in the art,progeny of an engineered polynucleotide or cell are typically stillreferred to as “engineered” even though the actual manipulation wasperformed on a prior entity. Furthermore, as will be appreciated bypersons of skill in the art, a variety of methodologies are availablethrough which “engineering” as described herein may be achieved. Forexample, in some embodiments, “engineering” may involve selection ordesign (e.g., of nucleic acid sequences, polypeptide sequences, cells,tissues, and/or organisms) through use of computer systems programmed toperform analysis or comparison, or otherwise to analyze, recommend,and/or select sequences, alterations, etc.). Alternatively, oradditionally, in some embodiments, “engineering” may involve use of invitro chemical synthesis methodologies and/or recombinant nucleic acidtechnologies such as, for example, nucleic acid amplification (e.g., viathe polymerase chain reaction) hybridization, mutation, transformation,transfection, etc., and/or any of a variety of controlled matingmethodologies. As will be appreciated by those skilled in the art, avariety of established such techniques (e.g., for recombinant DNA,oligonucleotide synthesis, and tissue culture and transformation (e.g.,electroporation, lipofection, etc.) are well known in the art anddescribed in various general and more specific references that are citedand/or discussed throughout the present specification. See e.g.,Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989 andPrinciples of Gene Manipulation: An Introduction to GeneticManipulation, 5th Ed., ed. By Old, R. W. and S. B. Primrose, BlackwellScience, Inc., 1994, incorporated herein by reference in theirentireties.

Functional: as used herein, refers to a form or fragment of an entity(e.g., a gene or gene segment) that exhibits a particular property(e.g., forms part of a coding sequence) and/or activity. For example, inthe context of immunoglobulins, variable regions are encoded by uniquegene segments (i.e., V, D and/or J) that are assembled (or recombined)to form functional coding sequences. When present in the genome, genesegments are organized in clusters, although variations do occur. A“functional” gene segment is a gene segment represented in an expressedsequence (i.e., a variable region) for which the corresponding genomicDNA has been isolated (i.e., cloned) and identified by sequence. Someimmunoglobulin gene segment sequences contain open reading frames andare considered functional although not represented in an expressedrepertoire, while other immunoglobulin gene segment sequences containmutations (e.g., point mutations, insertions, deletions, etc.) resultingin a stop codon and/or truncated sequence which subsequently render(s)such gene segment sequences unable to perform the property/ies and/oractivity/ies associated with a non-mutated sequence(s). Such sequencesare not represented in expressed sequences and, therefore, categorizedas pseudogenes.

Gene: as used herein, refers to a DNA sequence in a chromosome thatcodes for a product (e.g., an RNA product and/or a polypeptide product).In some embodiments, a gene includes coding sequence (i.e., sequencethat encodes a particular product). In some embodiments, a gene includesnon-coding sequence. In some particular embodiments, a gene may includeboth coding (e.g., exonic) and non-coding (e.g., intronic) sequence. Insome embodiments, a gene may include one or more regulatory sequences(e.g., promoters, enhancers, etc.) and/or intron sequences that, forexample, may control or impact one or more aspects of gene expression(e.g., cell-type-specific expression, inducible expression, etc.). Forthe purpose of clarity, we note that, as used in the present disclosure,the term “gene” generally refers to a portion of a nucleic acid thatencodes a polypeptide or fragment thereof; the term may optionallyencompass regulatory sequences, as will be clear from context to thoseof ordinary skill in the art. This definition is not intended to excludeapplication of the term “gene” to non-protein-coding expression unitsbut rather to clarify that, in most cases, the term as used in thisdocument refers to a polypeptide-coding nucleic acid.

Heterologous: as used herein, refers to an agent or entity from adifferent source. For example, when used in reference to a polypeptide,gene, or gene product present in a particular cell or organism, the termclarifies that the relevant polypeptide, gene, or gene product: 1) wasengineered by the hand of man; 2) was introduced into the cell ororganism (or a precursor thereof) through the hand of man (e.g., viagenetic engineering); and/or 3) is not naturally produced by or presentin the relevant cell or organism (e.g., the relevant cell type ororganism type). “Heterologous” also includes a polypeptide, gene or geneproduct that is normally present in a particular native cell ororganism, but has been altered or modified, for example, by mutation orplacement under the control of non-naturally associated and, in someembodiments, non-endogenous regulatory elements (e.g., a promoter).

Host cell: as used herein, refers to a cell into which a nucleic acid orprotein has been introduced. Persons of skill upon reading thisdisclosure will understand that such terms refer not only to theparticular subject cell, but also is used to refer to the progeny ofsuch a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the phrase “host cell.” In someembodiments, a host cell is or comprises a prokaryotic or eukaryoticcell. In general, a host cell is any cell that is suitable for receivingand/or producing a heterologous nucleic acid or protein, regardless ofthe Kingdom of life to which the cell is designated. Exemplary cellsinclude those of prokaryotes and eukaryotes (single-cell ormultiple-cell), bacterial cells (e.g., strains of Escherichia coli,Bacillus spp., Streptomyces spp., etc.), mycobacteria cells, fungalcells, yeast cells (e.g., Saccharomyces cerevisiae, Schizosaccharomycespombe, Pichia pastoris, Pichia methanolica, etc.), plant cells, insectcells (e.g., SF-9, SF-21, baculovirus-infected insect cells,Trichoplusia ni, etc.), non-human animal cells, human cells, or cellfusions such as, for example, hybridomas or quadromas. In someembodiments, a cell is a human, monkey, ape, hamster, rat, or mousecell. In some embodiments, a cell is eukaryotic and is selected from thefollowing cells: CHO (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g.,COS-7), retinal cell, Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR293, MDCK, HaK, BHK), HeLa, HepG2, W138, MRC 5, Colo205, HB 8065, HL-60,(e.g., BHK21), Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cell,C127 cell, SP2/0, NS-0, MMT 060562, Sertoli cell, BRL 3A cell, HT1080cell, myeloma cell, tumor cell, and a cell line derived from anaforementioned cell. In some embodiments, a cell comprises one or moreviral genes, e.g., a retinal cell that expresses a viral gene (e.g., aPER.C6® cell). In some embodiments, a host cell is or comprises anisolated cell. In some embodiments, a host cell is part of a tissue. Insome embodiments, a host cell is part of an organism.

Identity: as used herein in connection with a comparison of sequences,refers to identity as determined by a number of different algorithmsknown in the art that can be used to measure nucleotide and/or aminoacid sequence identity. In some embodiments, identities as describedherein are determined using a ClustalW v. 1.83 (slow) alignmentemploying an open gap penalty of 10.0, an extend gap penalty of 0.1, andusing a Gonnet similarity matrix (MACVECTOR™ 10.0.2, MacVector Inc.,2008).

In place of: as used herein, refers to a positional substitution inwhich a first nucleic acid sequence is located at the position of asecond nucleic acid sequence in a chromosome (e.g., where the secondnucleic acid sequence was previously (e.g., originally) located in achromosome, e.g., at the endogenous locus of the second nucleic acidsequence). The phrase “in place of” does not require that the secondnucleic acid sequence be removed from, e.g., a locus or chromosome. Insome embodiments, the second nucleic acid sequence and the first nucleicacid sequence are comparable to one another in that, for example, thefirst and second sequences are homologous to one another, containcorresponding elements (e.g., protein-coding elements, regulatoryelements, etc.), and/or have similar or identical sequences. In someembodiments, a first and/or second nucleic acid sequence includes one ormore of a promoter, an enhancer, a splice donor site, a splice acceptorsite, an intron, an exon, an untranslated region (UTR); in someembodiments, a first and/or second nucleic acid sequence includes one ormore coding sequences. In some embodiments, a first nucleic acidsequence is a homolog or variant (e.g., mutant) of the second nucleicacid sequence. In some embodiments, a first nucleic acid sequence is anortholog or homolog of the second sequence. In some embodiments, a firstnucleic acid sequence is or comprises a human nucleic acid sequence. Insome embodiments, including where the first nucleic acid sequence is orcomprises a human nucleic acid sequence, the second nucleic acidsequence is or comprises a rodent sequence (e.g., a mouse or ratsequence). In some embodiments, including where the first nucleic acidsequence is or comprises a human nucleic acid sequence, the secondnucleic acid sequence is or comprises a human sequence. In someembodiments, a first nucleic acid sequence is a variant or mutant (i.e.,a sequence that contains one or more sequence differences, e.g.,substitutions, as compared to the second sequence) of the secondsequence. The nucleic acid sequence so placed may include one or moreregulatory sequences that are part of source nucleic acid sequence usedto obtain the sequence so placed (e.g., promoters, enhancers, 5′- or3′-untranslated regions, etc.). For example, in various embodiments, afirst nucleic acid sequence is a substitution of an endogenous sequencewith a heterologous sequence that results in the production of a geneproduct from the nucleic acid sequence so placed (comprising theheterologous sequence), but not expression of the endogenous sequence; afirst nucleic acid sequence is of an endogenous genomic sequence with anucleic acid sequence that encodes a polypeptide that has a similarfunction as a polypeptide encoded by the endogenous sequence (e.g., theendogenous genomic sequence encodes a non-human variable regionpolypeptide, in whole or in part, and the DNA fragment encodes one ormore human variable region polypeptides, in whole or in part). Invarious embodiments, a human immunoglobulin gene segment or fragmentthereof is in place of an endogenous non-human immunoglobulin genesegment or fragment.

In vitro: as used herein refers to events that occur in an artificialenvironment, e.g., in a test tube or reaction vessel, in cell culture,etc., rather than within a multi-cellular organism.

In vivo: as used herein refers to events that occur within amulti-cellular organism, such as a human and/or a non-human animal. Inthe context of cell-based systems, the term may be used to refer toevents that occur within a living cell (as opposed to, for example, invitro systems).

Isolated: as used herein, refers to a substance and/or entity that hasbeen (1) separated from at least some of the components with which itwas associated when initially produced (whether in nature and/or in anexperimental setting), and/or (2) designed, produced, prepared, and/ormanufactured by the hand of man. Isolated substances and/or entities maybe separated from about 10%, about 20%, about 30%, about 40%, about 50%,about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,or more than about 99% of the other components with which they wereinitially associated. In some embodiments, isolated agents are separatedfrom 10% to 100%, 15%-100%, 20%-100%, 25%-100%, 30%-100%, 35%-100%,40%-100%, 45%-100%, 50%-100%, 55%-100%, 60%-100%, 65%-100%, 70%-100%,75%-100%, 80%-100%, 85%-100%, 90%-100%, 95%-100%, 96%-100%, 97%-100%,98%-100%, or 99%-100% of the other components with which they wereinitially associated. In some embodiments, isolated agents are separatedfrom 10% to 100%, 10%-99%, 10%-98%, 10%-97%, 10%-96%, 10%-95%, 10%-90%,10%-85%, 10%-80%, 10%-75%, 10%-70%, 10%-65%, 10%-60%, 10%-55%, 10%-50%,10%-45%, 10%-40%, 10%-35%, 10%-30%, 10%-25%, 10%-20%, or 10%-15% of theother components with which they were initially associated. In someembodiments, isolated agents are separated from 11% to 99%, 12%-98%,13%-97%, 14%-96%, 15%-95%, 20%-90%, 25%-85%, 30%-80%, 35%-75%, 40%-70%,45%-65%, 50%-60%, or 55%-60% of the other components with which theywere initially associated. In some embodiments, isolated agents areabout 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more thanabout 99% pure. In some embodiments, isolated agents are 80%-99%,85%-99%, 90%-99%, 95%-99%, 96%-99%, 97%-99%, or 98%-99% pure. In someembodiments, isolated agents are 80%-99%, 80%-98%, 80%-97%, 80%-96%,80%-95%, 80%-90%, or 80%-85% pure. In some embodiments, isolated agentsare 85%-98%, 90%-97%, or 95%-96% pure. In some embodiments, a substanceis “pure” if it is substantially free of other components. In someembodiments, as will be understood by those skilled in the art, asubstance may still be considered “isolated” or even “pure”, afterhaving been combined with certain other components such as, for example,one or more carriers or excipients (e.g., buffer, solvent, water, etc.);in such embodiments, percent isolation or purity of the substance iscalculated without including such carriers or excipients. To give butone example, in some embodiments, a biological polymer such as apolypeptide or polynucleotide that occurs in nature is considered to be“isolated” when: a) by virtue of its origin or source of derivation isnot associated with some or all of the components that accompany it inits native state in nature; b) it is substantially free of otherpolypeptides or nucleic acids of the same species from the species thatproduces it in nature; or c) is expressed by or is otherwise inassociation with components from a cell or other expression system thatis not of the species that produces it in nature. Thus, for instance, insome embodiments, a polypeptide that is chemically synthesized, or issynthesized in a cellular system different from that which produces itin nature, is considered to be an “isolated” polypeptide. Alternatively,or additionally, in some embodiments, a polypeptide that has beensubjected to one or more purification techniques may be considered to bean “isolated” polypeptide to the extent that it has been separated fromother components: a) with which it is associated in nature; and/or b)with which it was associated when initially produced.

Locus or loci: as used herein, refers to a location(s) of a gene (orsignificant sequence), DNA sequence, polypeptide-encoding sequence, orposition on a chromosome of the genome of an organism. For example, an“immunoglobulin locus” may refer to the location of an immunoglobulingene segment (e.g., V, D, J or C), immunoglobulin gene segment DNAsequence, immunoglobulin gene segment-encoding sequence, orimmunoglobulin gene segment position on a chromosome of the genome of anorganism that has been identified as to where such a sequence resides.An “immunoglobulin locus” may comprise a regulatory element of animmunoglobulin gene segment, including, but not limited to, an enhancer,a promoter, 5′ and/or 3′ regulatory sequence or region, or a combinationthereof. An “immunoglobulin locus” may comprise intergenic DNA, e.g.,DNA that normally resides or appears between gene segments in awild-type locus. Persons of ordinary skill in the art will appreciatethat chromosomes may, in some embodiments, contain hundreds or eventhousands of genes and demonstrate physical co-localization of similargenetic loci when comparing between different species. Such genetic locican be described as having shared synteny.

Naturally appears: as used herein in reference to a biological element(e.g., a nucleic acid sequence) means that the biological element can befound in a specified context and/or location, absent engineering (e.g.,genetic engineering), in a cell or organism (e.g., an animal). In otherwords, a sequence that naturally appears in a specified context and/orlocation is not in the specified context and/or location as the resultof engineering (e.g., genetic engineering). For example, a sequence thatnaturally appears adjacent to a human Jκ 1 gene segment in an endogenoushuman immunoglobulin kappa light chain locus is a sequence that can befound adjacent to a human Jκ1 gene segment in an endogenous humanimmunoglobulin kappa light chain locus, absent genetic engineering, in ahuman. In some embodiments, a sequence can be obtained, derived, and/orisolated from where it naturally appears in a cell or organism. In someembodiments, a cell or organism is not a direct source of a sequencethat naturally appears in the cell or organism. For example, acorresponding sequence in a cell or organism could be identified andthen produced or replicated by mechanisms known in the art.

Non-human animal: as used herein, refers to any vertebrate organism thatis not a human. In some embodiments, a non-human animal is a cyclostome,a bony fish, a cartilaginous fish (e.g., a shark or a ray), anamphibian, a reptile, a mammal, and a bird. In some embodiments, anon-human animal is a mammal. In some embodiments, a non-human mammal isa primate, a goat, a sheep, a pig, a dog, a cow, or a rodent. In someembodiments, a non-human animal is a rodent such as a rat or a mouse.

Nucleic acid: as used herein, refers to any compound and/or substancethat is or can be incorporated into an oligonucleotide chain. In someembodiments, a “nucleic acid” is a compound and/or substance that is orcan be incorporated into an oligonucleotide chain via a phosphodiesterlinkage. As will be clear from context, in some embodiments, “nucleicacid” refers to individual nucleic acid residues (e.g., nucleotidesand/or nucleosides); in some embodiments, “nucleic acid” refers to anoligonucleotide chain comprising individual nucleic acid residues. Insome embodiments, a “nucleic acid” is or comprises RNA; in someembodiments, a “nucleic acid” is or comprises DNA. In some embodiments,a “nucleic acid” is, comprises, or consists of one or more naturalnucleic acid residues. In some embodiments, a “nucleic acid” is,comprises, or consists of one or more nucleic acid analogs. In someembodiments, a nucleic acid analog differs from a “nucleic acid” in thatit does not utilize a phosphodiester backbone. For example, in someembodiments, a “nucleic acid” is, comprises, or consists of one or more“peptide nucleic acids”, which are known in the art and have peptidebonds instead of phosphodiester bonds in the backbone. Alternatively, oradditionally, in some embodiments, a “nucleic acid” has one or morephosphorothioate and/or 5′-N-phosphoramidite linkages rather thanphosphodiester bonds. In some embodiments, a “nucleic acid” is,comprises, or consists of one or more natural nucleosides (e.g.,adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine,deoxythymidine, deoxyguanosine, and deoxycytidine). In some embodiments,a “nucleic acid” is, comprises, or consists of one or more nucleosideanalogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine,pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine,C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine,C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine,7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine,O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalatedbases, and combinations thereof). In some embodiments, a “nucleic acid”comprises one or more modified sugars (e.g., 2′-fluororibose, ribose,2′-deoxyribose, arabinose, and hexose) as compared with those in naturalnucleic acids. In some embodiments, a “nucleic acid” has a nucleotidesequence that encodes a functional gene product such as an RNA orpolypeptide. In some embodiments, a “nucleic acid” includes one or moreintrons. In some embodiments, a “nucleic acid” includes one or moreexons. In some embodiments, a “nucleic acid” is prepared by one or moreof isolation from a natural source, enzymatic synthesis bypolymerization based on a complementary template (in vivo or in vitro),reproduction in a recombinant cell or system, and chemical synthesis. Insome embodiments, a “nucleic acid” is at least, e.g., but not limitedto, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500,600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000or more residues long. In some embodiments, a “nucleic acid” is singlestranded; in some embodiments, a “nucleic acid” is double stranded. Insome embodiments, a “nucleic acid” has a nucleotide sequence comprisingat least one element that encodes, or is the complement of a sequencethat encodes, a polypeptide. In some embodiments, a “nucleic acid” hasenzymatic activity.

Operably linked: as used herein, refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. A control sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under conditions compatible with the controlsequences. “Operably linked” sequences include both expression controlsequences that are contiguous with a gene of interest and expressioncontrol sequences that act in trans or at a distance to control a geneof interest (or sequence of interest). The term “expression controlsequence” includes polynucleotide sequences, which are necessary toaffect the expression and processing of coding sequences to which theyare ligated. “Expression control sequences” include: appropriatetranscription initiation, termination, promoter and enhancer sequences;efficient RNA processing signals such as splicing and polyadenylationsignals; sequences that stabilize cytoplasmic mRNA; sequences thatenhance translation efficiency (i.e., Kozak consensus sequence);sequences that enhance polypeptide stability; and when desired,sequences that enhance polypeptide secretion. The nature of such controlsequences differs depending upon the host organism. For example, inprokaryotes, such control sequences generally include promoter,ribosomal binding site and transcription termination sequence, while ineukaryotes typically such control sequences include promoters andtranscription termination sequence. The term “control sequences” isintended to include components whose presence is essential forexpression and processing, and can also include additional componentswhose presence is advantageous, for example, leader sequences and fusionpartner sequences.

Physiological conditions: as used herein, refers to its art-understoodmeaning referencing conditions under which cells or organisms liveand/or reproduce. In some embodiments, the term includes conditions ofthe external or internal milieu that may occur in nature for an organismor cell system. In some embodiments, physiological conditions are thoseconditions present within the body of a human or non-human animal,especially those conditions present at and/or within a surgical site.Physiological conditions typically include, e.g., a temperature range of20-40° C., atmospheric pressure of 1, pH of 6-8, glucose concentrationof 1-20 mM, oxygen concentration at atmospheric levels, and gravity asit is encountered on earth. In some embodiments, conditions in alaboratory are manipulated and/or maintained at physiologic conditions.In some embodiments, physiological conditions are encountered in anorganism.

Polypeptide: as used herein, refers to any polymeric chain of aminoacids. In some embodiments, a polypeptide has an amino acid sequencethat occurs in nature. In some embodiments, a polypeptide has an aminoacid sequence that does not occur in nature. In some embodiments, apolypeptide has an amino acid sequence that contains portions that occurin nature separately from one another (i.e., from two or more differentorganisms, for example, human and non-human portions). In someembodiments, a polypeptide has an amino acid sequence that is engineeredin that it is designed and/or produced through action of the hand ofman. In some embodiments, a polypeptide has an amino acid sequenceencoded by a sequence that does not occur in nature (e.g., a sequencethat is engineered in that it is designed and/or produced through actionof the hand of man to encode said polypeptide).

Recombinant: as used herein, refers to polypeptides that are designed,engineered, prepared, expressed, created or isolated by recombinantmeans, such as polypeptides expressed using a recombinant expressionvector transfected into a host cell, polypeptides isolated from arecombinant, combinatorial human polypeptide library (Hoogenboom, H. R.,1997, TIB Tech. 15:62-70; Azzazy, H. and W. E. Highsmith, 2002, Clin.Biochem. 35:425-45; Gavilondo, J. V. and J. W. Larrick, 2002,BioTechniques 29:128-45; Hoogenboom H., and P. Chames, 2000, Immunol.Today 21:371-8, incorporated herein by reference in their entireties),antibodies isolated from an animal (e.g., a mouse) that has beengenetically engineered to include human immunoglobulin genes (see e.g.,Taylor, L. D. et al., 1992, Nucl. Acids Res. 20:6287-95; Kellermann,S-A. and L. L. Green, 2002, Curr. Opin. Biotechnol. 13:593-7; Little, M.et al., 2000, Immunol. Today 21:364-70; Osborn, M. J. et al., 2013, J.Immunol. 190:1481-90; Lee, E-C. et al., 2014, Nat. Biotech.32(4):356-63; Macdonald, L. E. et al., 2014, Proc. Natl. Acad. Sci.U.S.A. 111(14):5147-52; Murphy, A. J. et al., 2014, Proc. Natl. Acad.Sci. U.S.A. 111(14):5153-8, each of which is incorporated herein byreference in its entirety) or polypeptides prepared, expressed, createdor isolated by any other means that involves splicing selected sequenceelements to one another. In some embodiments, one or more of suchselected sequence elements is found in nature. In some embodiments, oneor more of such selected sequence elements is designed in silico. Insome embodiments, one or more such selected sequence elements resultfrom mutagenesis (e.g., in vivo or in vitro) of a known sequenceelement, e.g., from a natural or synthetic (e.g., man-made) source. Forexample, in some embodiments, a recombinant polypeptide is comprised ofsequences found in the genome of a source organism of interest (e.g.,human, mouse, etc.). In some embodiments, a recombinant polypeptide hasan amino acid sequence that resulted from mutagenesis (e.g., in vitro orin vivo, for example, in a non-human animal), so that the amino acidsequences of the recombinant polypeptides are sequences that, whileoriginating from and related to polypeptides sequences, may notnaturally exist within the genome of a non-human animal in vivo.

Reference: as used herein, refers to a standard or control agent,animal, cohort, individual, population, sample, sequence or valueagainst which an agent, animal, cohort, individual, population, sample,sequence or value of interest is compared. In some embodiments, areference agent, animal, cohort, individual, population, sample,sequence or value is tested and/or determined substantiallysimultaneously with the testing or determination of an agent, animal,cohort, individual, population, sample, sequence or value of interest.In some embodiments, a reference agent, animal, cohort, individual,population, sample, sequence or value is a historical reference,optionally embodied in a tangible medium. In some embodiments, areference may refer to a control. A “reference” also includes a“reference animal.” A “reference animal” may have a modification asdescribed herein, a modification that is different as described hereinor no modification (i.e., a wild-type animal). Typically, as would beunderstood by persons of skill in the art, a reference agent, animal,cohort, individual, population, sample, sequence or value is determinedor characterized under conditions comparable to those utilized todetermine or characterize an agent, animal (e.g., a mammal), cohort,individual, population, sample, sequence or value of interest.

Replacement: as used herein, refers to a process through which a“replaced” nucleic acid sequence (e.g., a gene) found in a host locus(e.g., in a genome) is removed from that locus, and a different,“replacement” nucleic acid is located in its place. In some embodiments,the replaced nucleic acid sequence and the replacement nucleic acidsequences are comparable to one another in that, for example, they arehomologous to one another, contain corresponding elements (e.g.,protein-coding elements, regulatory elements, etc.), and/or have similaror identical sequences. In some embodiments, a replaced nucleic acidsequence includes one or more of a promoter, an enhancer, a splice donorsite, a splice acceptor site, an intron, an exon, an untranslated region(UTR); in some embodiments, a replacement nucleic acid sequence includesone or more coding sequences. In some embodiments, a replacement nucleicacid sequence is a homolog or variant (e.g., mutant) of the replacednucleic acid sequence. In some embodiments, a replacement nucleic acidsequence is an ortholog or homolog of the replaced sequence. In someembodiments, a replacement nucleic acid sequence is or comprises a humannucleic acid sequence. In some embodiments, including where thereplacement nucleic acid sequence is or comprises a human nucleic acidsequence, the replaced nucleic acid sequence is or comprises a rodentsequence (e.g., a mouse or rat sequence). In some embodiments, includingwhere the replacement nucleic acid sequence is or comprises a humannucleic acid sequence, the replaced nucleic acid sequence is orcomprises a human sequence. In some embodiments, a replacement nucleicacid sequence is a variant or mutant (i.e., a sequence that contains oneor more sequence differences, e.g., substitutions, as compared to thereplaced sequence) of the replaced sequence. The nucleic acid sequenceso placed may include one or more regulatory sequences that are part ofsource nucleic acid sequence used to obtain the sequence so placed(e.g., promoters, enhancers, 5′- or 3′-untranslated regions, etc.). Forexample, in various embodiments, a replacement is a substitution of anendogenous sequence with a heterologous sequence that results in theproduction of a gene product from the nucleic acid sequence so placed(comprising the heterologous sequence), but not expression of theendogenous sequence; a replacement is of an endogenous genomic sequencewith a nucleic acid sequence that encodes a polypeptide that has asimilar function as a polypeptide encoded by the endogenous sequence(e.g., the endogenous genomic sequence encodes a non-human variableregion polypeptide, in whole or in part, and the DNA fragment encodesone or more human variable region polypeptides, in whole or in part). Invarious embodiments, an endogenous non-human immunoglobulin gene segmentor fragment thereof is replaced with a human immunoglobulin gene segmentor fragment thereof.

Substantially: as used herein, refers to the qualitative condition ofexhibiting total or near-total extent or degree of a characteristic orproperty of interest. One of ordinary skill in the biological arts willunderstand that biological and chemical phenomena rarely, if ever, go tocompletion and/or proceed to completeness or achieve or avoid anabsolute result. The term “substantially” is therefore used herein tocapture the potential lack of completeness inherent in many biologicaland chemical phenomena.

Substantial similarity: as used herein, refers to a comparison betweenamino acid or nucleic acid sequences. As will be appreciated by those ofordinary skill in the art, two sequences are generally considered to be“substantially similar” if they contain similar residues (e.g., aminoacids or nucleotides) in corresponding positions. As is understood inthe art, while similar residues may be identical residues (see alsoSubstantial Identity, below), similar residues may also be non-identicalresidues with appropriately comparable structural and/or functionalcharacteristics. For example, as is well known by those of ordinaryskill in the art, certain amino acids are typically classified as“hydrophobic” or “hydrophilic” amino acids, and/or as having “polar” or“non-polar” side chains. Substitution of one amino acid for another ofthe same type may often be considered a “conservative” substitution.Typical amino acid categorizations are summarized in the table below.

Alanine Ala A Nonpolar Neutral 1.8 Arginine Arg R Polar Positive −4.5Asparagine Asn N Polar Neutral −3.5 Aspartic acid Asp D Polar Negative−3.5 Cysteine Cys C Nonpolar Neutral 2.5 Glutamic acid Glu E PolarNegative −3.5 Glutamine Gln Q Polar Neutral −3.5 Glycine Gly G NonpolarNeutral −0.4 Histidine His H Polar Positive −3.2 Isoleucine Ile INonpolar Neutral 4.5 Leucine Leu L Nonpolar Neutral 3.8 Lysine Lys KPolar Positive −3.9 Methionine Met M Nonpolar Neutral 1.9 PhenylalaninePhe F Nonpolar Neutral 2.8 Proline Pro P Nonpolar Neutral −1.6 SerineSer S Polar Neutral −0.8 Threonine Thr T Polar Neutral −0.7 TryptophanTrp W Nonpolar Neutral −0.9 Tyrosine Tyr Y Polar Neutral −1.3 Valine ValV Nonpolar Neutral 4.2 Ambiguous Amino Acids 3-Letter 1-LetterAsparagine or aspartic acid Asx B Glutamine or glutamic acid Glx ZLeucine or Isoleucine Xle J Unspecified or unknown amino acid Xaa X

As is well known in this art, amino acid or nucleic acid sequences maybe compared using any of a variety of algorithms, including thoseavailable in commercial computer programs such as BLASTN for nucleotidesequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acidsequences. Exemplary such programs are described in Altschul, S. F. etal., 1990, J. Mol. Biol., 215(3): 403-10; Altschul, S. F. et al., 1996,Meth. Enzymol. 266:460-80; Altschul, S. F. et al., 1997, Nucleic AcidsRes., 25:3389-402; Baxevanis, A. D. and B. F. F. Ouellette (eds.)Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins,Wiley, 1998; and Misener et al. (eds.) Bioinformatics Methods andProtocols, Methods in Molecular Biology, Vol. 132, Humana Press, 1998,incorporated herein by reference in their entireties. In addition toidentifying similar sequences, the programs mentioned above typicallyprovide an indication of the degree of similarity. In some embodiments,two sequences are considered to be substantially similar if at least,e.g., but not limited to, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more of their corresponding residues are similar (e.g.,identical or include a conservative substitution) over a relevantstretch of residues. In some embodiments, the relevant stretch is acomplete sequence (e.g. a sequence of a gene, a gene segment, a sequenceencoding a domain, a polypeptide, or a domain). In some embodiments, therelevant stretch is at least 9, 10, 11, 12, 13, 14, 15, 16, 17 or moreresidues. In some embodiments, the relevant stretch is at least 10, 15,20, 25, 30, 35, 40, 45, 50, or more residues. In some embodiments, therelevant stretch includes contiguous residues along a complete sequence.In some embodiments, the relevant stretch includes discontinuousresidues along a complete sequence, for example, noncontiguous residuesbrought together by the folded conformation of a polypeptide or aportion thereof.

Substantial identity: as used herein, refers to a comparison betweenamino acid or nucleic acid sequences. As will be appreciated by those ofordinary skill in the art, two sequences are generally considered to be“substantially identical” if they contain identical residues (e.g.,amino acids or nucleotides) in corresponding positions. As is well-knownin this art, amino acid or nucleic acid sequences may be compared usingany of a variety of algorithms, including those available in commercialcomputer programs such as BLASTN for nucleotide sequences and BLASTP,gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplary suchprograms are described in Altschul, S. F. et al., 1990, J. Mol. Biol.,215(3): 403-10; Altschul, S. F. et al., 1996, Meth. Enzymol. 266:460-80;Altschul, S. F. et al., 1997, Nucleic Acids Res., 25:3389-402;Baxevanis, A. D. and B. F. F. Ouellette (eds.) Bioinformatics: APractical Guide to the Analysis of Genes and Proteins, Wiley, 1998; andMisener et al. (eds.) Bioinformatics Methods and Protocols, Methods inMolecular Biology, Vol. 132, Humana Press, 1998, each of which isincorporated herein by reference in its entirety. In addition toidentifying identical sequences, the programs mentioned above typicallyprovide an indication of the degree of identity. In some embodiments,two sequences are considered to be substantially identical if at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more oftheir corresponding residues are identical over a relevant stretch ofresidues. In some embodiments, a relevant stretch of residues is acomplete sequence. In some embodiments, a relevant stretch of residuesis, e.g., but not limited to, at least 10, 15, 20, 25, 30, 35, 40, 45,50, or more residues.

Targeting construct or targeting vector: as used herein, refers to apolynucleotide molecule that comprises a targeting region. A targetingregion comprises a sequence that is identical or substantially identicalto a sequence in a target cell, tissue or animal and provides forintegration of the targeting construct into a position within the genomeof the cell, tissue or animal via homologous recombination. Targetingregions that target using site-specific recombinase recognition sites(e.g., IoxP or Frt sites) are also included and described herein. Insome embodiments, a targeting construct as described herein furthercomprises a nucleic acid sequence or gene of particular interest, aselectable marker, control and/or regulatory sequences, and othernucleic acid sequences that allow for recombination mediated throughexogenous addition of proteins that aid in or facilitate recombinationinvolving such sequences. In some embodiments, a targeting construct asdescribed herein further comprises a gene of interest in whole or inpart, wherein the gene of interest is a heterologous gene that encodes apolypeptide, in whole or in part, that has a similar function as aprotein encoded by an endogenous sequence. In some embodiments, atargeting construct as described herein further comprises a humanizedgene of interest, in whole or in part, wherein the humanized gene ofinterest encodes a polypeptide, in whole or in part, that has a similarfunction as a polypeptide encoded by an endogenous sequence. In someembodiments, a targeting construct (or targeting vector) may comprise anucleic acid sequence manipulated by the hand of man. For example, insome embodiments, a targeting construct (or targeting vector) may beconstructed to contain an engineered or recombinant polynucleotide thatcontains two or more sequences that are not linked together in thatorder in nature yet manipulated by the hand of man to be directly linkedto one another in the engineered or recombinant polynucleotide.

Transgene or transgene construct: as used herein, refers to a nucleicacid sequence (encoding e.g., a polypeptide of interest, in whole or inpart) that has been introduced into a cell by the hand of man such as bythe methods described herein. A transgene could be partly or entirelyheterologous, i.e., foreign, to the genetically engineered animal orcell into which it is introduced. A transgene can include one or moretranscriptional regulatory sequences and any other nucleic acid, such asintrons or promoters, which may be necessary for expression of aselected nucleic acid sequence.

Genetically modified non-human animal or genetically engineerednon-human animal: are used interchangeably herein and refer to anynon-naturally occurring non-human animal in which one or more of thecells of the non-human animal contain heterologous nucleic acid and/orgene encoding a polypeptide of interest, in whole or in part. Forexample, in some embodiments, a “genetically modified non-human animal”or “genetically engineered non-human animal” refers to non-human animalthat contains a transgene or transgene construct as described herein. Insome embodiments, a heterologous nucleic acid and/or gene is introducedinto the cell, directly or indirectly by introduction into a precursorcell, by way of deliberate genetic manipulation, such as bymicroinjection or by infection with a recombinant virus. The termgenetic manipulation does not include classic breeding techniques, butrather is directed to introduction of recombinant DNA molecule(s). Thismolecule may be integrated within a chromosome, or it may beextrachromosomally replicating DNA. The phrases “genetically modifiednon-human animal” or “genetically engineered non-human animal” refers toanimals that are heterozygous or homozygous for a heterologous nucleicacid and/or gene, and/or animals that have single or multi-copies of aheterologous nucleic acid and/or gene.

Vector: as used herein, refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it is associated. In someembodiment, vectors are capable of extra-chromosomal replication and/orexpression of nucleic acids to which they are linked in a host cell suchas a eukaryotic and/or prokaryotic cell. Vectors capable of directingthe expression of operably linked genes are referred to herein as“expression vectors.”

Wild-type: as used herein, refers to an entity having a structure and/oractivity as found in nature in a “normal” (as contrasted with mutant,diseased, altered, etc.) state or context. Those of ordinary skill inthe art will appreciate that wild-type genes and polypeptides oftenexist in multiple different forms (e.g., alleles).

DETAILED DESCRIPTION

The present disclosure provides, among other things, engineerednon-human animals having heterologous genetic material encoding human Vλdomains, which heterologous genetic material comprises human Vλ and Jλgene sequences (i.e., gene segments) and other human sequences thatprovide for proper rearrangement (e.g., recombination signal sequence(RSS)) and expression of antibodies having Igλ light chains that includea human portion and a non-human portion, or antibodies having Igλ lightchains that are fully human. For example, in various embodiments, when ahuman gene segment is present in a genome of an engineered non-humananimal, the corresponding recombination signal sequence(s) can also bepresent (e.g., Vλ RSS with Vλ gene segment, Jλ RSS with Jλ gene segment,Vκ RSS with Vκ gene segment, Jκ RSS with Jκ gene segment, etc.). Invarious embodiments, provided engineered non-human animals containheterologous genetic material that is inserted in such a way so thatantibodies containing light chains that have a human Vλ domain and anon-human or human CX domain are expressed in the antibody repertoire ofthe non-human animal. Further, provided engineered non-human animalscontain heterologous genetic material that is inserted in such a way sothat antibodies containing light chains that have a human Vλ domain anda non-human or human CX domain are expressed from engineered Igκ lightchain loci that include human and non-human Igλ gene sequences (e.g.,gene segments) and, in some embodiments, human Igκ light chainsequences, in the germline genome of the non-human animal.

Without wishing to be bound by any particular theory, it is contemplatedthat non-human animals as described herein provide an improved in vivosystem that exploits the expression of antibodies containing human Vλdomains for the production of therapeutic antibodies. It is alsocontemplated that non-human animals as described herein, in someembodiments, provide alternate engineered forms of light chain loci(e.g., Igκ light chain loci) that contain heterologous genetic materialfor the development of human antibody-based therapeutics (e.g., humanmonoclonal antibodies, multi-specific binding agents, scFvs, fusionpolypeptides, etc.) to disease targets that are associated with biasedantibody responses (e.g., antibody responses characterized by anoverwhelming proportion of either K or λ light chains). Thus, providednon-human animals are particularly useful for the development of humanantibodies and human antibody-based molecules (e.g., multi-specificbinding agents, scFvs, fusion polypeptides, etc.) against targetsassociated with poor immunogenicity (e.g., viruses) due, in part, toskewed antibody repertoires and/or responses.

The present disclosure describes, among other things, an immunoglobulinκ light chain locus that includes one or more human Vλ gene segments,one or more human Jλ gene segments, and a Cλ gene. Such a locus isreferred to as a “lambda in kappa” locus or “LiK”.

In particular, the present disclosure describes the production of anon-human animal (e.g., a rodent) having a germline genome that containsan engineered Igκ light chain locus that is, in some embodiments,characterized by the introduction of a plurality of human Vλ and Jλ genesegments and introduction of a non-human or human Cλ gene in the placeof a non-human Cκ gene, so that said plurality of human Vλ and Jλ genesegments are operably linked to said non-human or human Cλ gene. Asdescribed herein, the production of such an engineered Igκ light chainlocus results in the expression of antibodies that contain light chainsthat include a human Vλ domain and a non-human or human Cλ domain fromsaid engineered Igκ light chain locus in the germline genome of thenon-human animal. In some embodiments, the germline genome of providednon-human animals comprises an Igκ light chain locus including human Igλlight chain sequences. In some embodiments, the germline genome ofprovided non-human animals comprises (i) an Igκ light chain locusincluding human Igλ light chain sequences, and (ii)(a) an Igκ lightchain locus including human Igλ light chain sequences or (ii)(b) an Igκlight chain locus including human Igκ light chain sequences. Thegermline genome of provided non-human animals, in some embodiments,comprises an Igλ light chain locus as described herein and furthercomprises (i) a humanized IgH locus or (ii) a humanized IgH locus andfunctionally silenced or otherwise rendered non-functional endogenousIgλ light chain locus. Provided non-human animals, as described herein,express antibody repertoires that contain Igλ light chains that includehuman Vλ domains.

In some embodiments, non-human animals as described herein contain humanIgλ light chain sequences within an Igκ light chain locus. In someembodiments, non-human animals as described herein contain human andnon-human Igλ light chain sequences within an Igκ light chain locus. Insome embodiments, non-human animals as described herein contain humanIgλ and human Igκ light chain sequences within an Igκ light chain locus.In some embodiments, non-human animals as described herein contain humanIgλ, human Igκ and murine Igκ and/or murine Igλ light chain sequenceswithin an Igκ light chain locus. In some embodiments, non-human animalsas described herein contain human Igλ light chain sequences, non-humanIgλ light chain sequences, human Igκ light chain sequences, non-humanIgκ light chain sequences, or combinations thereof within an Igκ lightchain locus. In many embodiments of non-human animals as describedherein, non-human sequences are or comprise murine sequences (e.g.,mouse or rat).

In some embodiments, Igκ and/or Igλ light chain sequences includeintergenic DNA that is of human and/or murine origin. In someembodiments, Igκ and/or Igλ light chain sequences include intergenic DNAthat is engineered and based on a source sequence that is of human ormurine origin. In some embodiments, said intergenic DNA is of the sameimmunoglobulin locus in which the intergenic DNA is so placed, inserted,positioned or engineered (e.g., Igκ intergenic DNA in an Igκ light chainlocus). In some embodiments, said intergenic DNA is of a differentimmunoglobulin locus in which the intergenic DNA is so placed, inserted,positioned or engineered (e.g., Igλ intergenic DNA in an Igκ light chainlocus). In some certain embodiments, non-human animals as describedherein contain an engineered Igκ light chain locus that containsintergenic DNA that includes Igκ light chain sequence(s), Igλ lightchain sequence(s) and/or combinations thereof.

In various embodiments, a humanized immunoglobulin heavy chain locuscontains at least one human V_(H), at least one human D_(H) and at leastone human J_(H) gene segment operably linked to to a non-humanimmunoglobulin heavy chain constant region (e.g., an endogenousnon-human immunoglobulin heavy chain constant region that includes oneor more immunoglobulin heavy chain constant region genes such as, forexample, IgM, IgD, IgG, IgE, IgA, etc.), e.g., a plurality of humanV_(H), D_(H) and J_(H) gene segments operably linked to a non-humanimmunoglobulin heavy chain constant region. In some embodiments,provided non-human animals have a germline genome that includes one ormore immunoglobulin loci depicted in the Drawings. Such engineerednon-human animals provide a source of human antibodies and humanantibody fragments, and provide an improved in vivo system suitable forexploiting human Vλ sequences for the production of human therapeuticantibodies.

As described in the Examples section below, non-human animals areprovided that have a genome that contains at least one of each humanheavy (i.e., V_(H), D_(H) and J_(H)) and light chain (e.g., Vλ and Jλ atthe endogenous kappa locus) variable region gene segments, e.g., aplurality of human heavy (i.e., V_(H), D_(H) and J_(H)) and light chain(e.g., Vλ and Jλ at the endogenous kappa locus) variable region genesegments, in the place of non-human variable region gene segments atendogenous immunoglobulin loci, and include human non-coding intergenicDNA between the human variable region gene segments. Such intergenic DNAincludes, for example, promoters, leader sequences and recombinationsignal sequences that allow for proper recombination and expression ofthe human gene segments in the context of variable domains ofantibodies. Persons of skill understand that non-human immunoglobulinloci also contain such non-coding intergenic DNA. Upon reading thisdisclosure, persons of skill will understand that other human ornon-human intergenic DNA can be employed in constructing such lociresulting in the same expression of human variable domains in thecontext of antibodies in the non-human animal. Such similar loci needonly contain the human coding sequences (i.e., exons) of the desiredhuman gene segments to achieve expression of antibodies that containhuman variable domains.

Various aspects of certain embodiments are described in detail in thefollowing sections, each of which can apply to any aspect or embodimentas described herein. The use of sections is not for limitation.

Antibody Repertoires in Non-Human Animals

Immunoglobulins (also called antibodies) are large (˜150 kD), Y-shapedglycoproteins that are produced by B cells of a host immune system toneutralize pathogens (e.g., viruses, bacteria, etc.). Eachimmunoglobulin (Ig) is composed of two identical heavy chains and twoidentical light chains, each of which has two structural components: avariable domain and a constant domain. The heavy and light chainvariable regions differ in antibodies produced by different B cells, butare the same for all antibodies produced by a single B cell or B cellclone. The heavy and light chain variable regions of each antibodytogether comprise the antigen-binding region (or antigen-binding site).Immunoglobulins can exist in different varieties that are referred to asisotypes or classes based on the heavy chain constant regions (ordomains) that they contain. The heavy chain constant region is identicalin all antibodies of the same isotype, but differs in antibodies ofdifferent isotypes. The table below summarizes the nine antibodyisotypes in mouse and human.

Mouse Human IgM IgM IgD IgD IgG1 IgG1 IgG2a IgG2 IgG2b IgG3 IgG2c IgG4IgG3 IgE IgE IgA1 IgA IgA2

Additional isotypes have been identified in other species. Isotypesconfer specialized biological properties on the antibody due to thedifferent structural characteristics among the different isotypes andare found in different locations (cells, tissues, etc.) within an animalbody. Initially, B cells produce IgM and IgD with identicalantigen-binding regions. Upon activation, B cells switch to differentisotypes by a process referred to as class switching, which involves achange of the constant region of the antibody produced by the B cellwhile the variable regions remain the same, thereby preserving antigenspecificity of the original antibody (B cell).

Two separate loci (Igκ and Igλ) contain the gene segments that, uponrearrangement, encode the light chains of antibodies, and exhibit bothallelic and isotypic exclusion. The expression ratios of κ⁺ to λ⁺ Bcells vary among species. For example, humans demonstrate a ratio ofabout 60:40 (κ:λ). In mice and rats, a ratio of 95:5 (κ:λ) is observed.Interestingly, the κ:λ ratio observed in cats (5:95) is opposite of miceand rats. Several studies have been conducted to elucidate the possiblereasons behind these observed ratios, and both the complexity of thelocus (i.e., number of gene segments, in particular, V gene segments)and the efficiency of gene segment rearrangement have been proposed asrationale. The human Igλ light chain locus extends over 1,000 kb andcontains approximately 70 Vλ gene segments (29 to 33 functional) andseven Jλ-Cλ gene segment pairs (four to five functional) organized intothree clusters (see, e.g., FIG. 1 of U.S. Pat. No. 9,006,511, which isincorporated herein by reference in its entirety). The majority of theobserved Vλ regions in the expressed antibody repertoire are encoded bygene segments contained within the most proximal cluster (referred to ascluster A). The mouse Igλ light chain locus is strikingly different thanthe human locus and, depending on the strain, contains only a few Vλ andJλ gene segments organized in two distinct gene clusters (see, e.g.,FIG. 2 of U.S. Pat. No. 9,006,511, which is incorporated herein byreference in its entirety).

Development of therapeutic antibodies for the treatment of various humandiseases has largely been centered on the creation of engineerednon-human animal lines, in particular, engineered rodent lines,harboring varying amounts of genetic material in their genomescorresponding to human immunoglobulin genes (reviewed in, e.g.,Bruggemann, M. et al., 2015, Arch. Immunol. Ther. Exp. 63:101-8, whichis incorporated herein by reference in its entirety). Initial efforts increating such genetically engineered rodent lines focused on integrationof portions of human immunoglobulin loci that could, by themselves,support recombination of gene segments and production of heavy and/orlight chains that were entirely human while having endogenousimmunoglobulin loci inactivated (see e.g., Bruggemann, M. et al., 1989,Proc. Nat. Acad. Sci. U.S.A. 86:67-09-13; Bruggemann, M. et al., 1991,Eur. J. Immunol. 21:1323-6; Taylor, L. D. et al., 1992, Nucl. Acids Res.20:6287-6295; Davies, N. P. et al., 1993, Biotechnol. 11:911-4; Green,L. L. et al., 1994, Nat. Genet. 7:13-21; Lonberg, N. et al., 1994,Nature 368:856-9; Taylor, L. D. et al., 1994, Int. Immunol. 6:579-91;Wagner, S. D. et al., 1994, Eur. J. Immunol. 24:2672-81; Fishwild, D. M.et al., 1996, Nat. Biotechnol. 14:845-51; Wagner, S. D. et al., 1996,Genomics 35:405-14; Mendez, M. J. et al., 1997, Nat. Genet. 15:146-56;Green, L. L. et al., 1998, J. Exp. Med. 188:483-95; Xian, J. et al.,1998, Transgenics 2:333-43; Little, M. et al., 2000, Immunol. Today21:364-70; Kellermann, S. A. and L. L. Green, 2002, Cur. Opin.Biotechnol. 13:593-7, each of which is incorporated by reference intheir entirety). In particular, some efforts have included integrationof human Igλ light chain sequences (see, e.g., U.S. Patent ApplicationPublication Nos. 2002/0088016 A1, 2003/0217373 A1 and 2011/0236378 A1;U.S. Pat. Nos. 6,998,514 and 7,435,871; Nicholson, I. C. et al., 1999,J. Immunol. 163:6898-906; Popov, A. V et al., 1999, J. Exp. Med.189(10): 1611-19, each of which is incorporated herein by reference inits entirety). Such efforts have focused on the random integration ofyeast artificial chromosomes containing human Vλ, Jλ and Cλ sequencesthereby creating mouse strains that express fully human Igλ light chains(i.e., human Vλ and Cλ domains). More recent efforts have employedsimilar strategies using constructs that also contain human Vλ, Jλ andCλ sequences (Osborn, M. J. et al., 2013, J. Immunol. 190:1481-90; Lee,E-C. et al., 2014, Nat. Biotech. 32(4):356-63, each of which isincorporated herein by reference in its entirety).

Yet other efforts have included the specific insertion of human Vλ andJλ gene segments into endogenous rodent Ig light chain loci (κ and λ) sothat said human Vλ and Jλ gene segments are operably linked toendogenous Ig light chain constant region genes (see, e.g., U.S. Pat.Nos. 9,006,511, 9,012,717, 9,029,628, 9,035,128, 9,066,502, 9,150,662and 9,163,092; all of which are incorporated herein by reference intheir entireties). In such animals, all of the human Vλ gene segmentsfrom clusters A and B and either one or four human Jλ gene segments wereinserted into endogenous Igκ and Igλ light chain loci. As a result,several different human Vλ and Jλ gene segments demonstrated properrearrangement at both engineered rodent Ig light chain loci to formfunctional light chains expressed in the rodent antibody repertoire,which light chains included human Vλ domains in the context of eitherendogenous Cκ and Cλ regions (see, e.g., Table 7 and FIGS. 11-13 of U.S.Pat. No. 9,006,511, which is incorporated herein by reference in itsentirety). In particular, mice having engineered Igκ light chain lociharboring human Vλ and Jλ gene segments demonstrated a human lambda toendogenous lambda ratio (as measured by IgCκ to IgCλ ratio) of about 1:1in the splenic compartment (see, e.g., Table 4 of U.S. Pat. No.9,006,511, which is incorporated herein by reference in its entirety).Indeed, both engineered mouse strains (i.e., engineered Igκ orengineered Igλ light chain loci) demonstrated that human Vλ domainscould be expressed from endogenous Ig light chain loci in rodents, whichnormally display a large bias in light chain expression (see above). Thepresent disclosure provides the recognition that alternate engineered Iglight chain locus structures can be produced to maximize usage of humanVλ and Jλ gene segments in antibody repertoires to therapeutic targetsin non-human animals, in particular, as compared to non-human animalsthat contain an Igλ light chain locus that lacks the complexity androbust quality (e.g., mice and rats) that is normally associated with ahuman Igλ light chain locus (i.e., such a locus that appears in a humancell). Such alternate engineered Ig light chain locus structures providethe capacity for unique antibody repertoires resulting from theirdesign.

The present disclosure exemplifies the successful production of anon-human animal whose germline genome contains an engineered endogenousIgκ light chain locus comprising a plurality of human Vλ and Jλ genesegments in operable linkage to a non-human or human Igλ light chainconstant region gene, which non-human or human Igλ light chain constantregion gene is inserted in the place of a non-human Igκ light chainconstant region gene of the endogenous Igx light chain locus. Inparticular, the present disclosure specifically demonstrates thesuccessful production of (1) an engineered non-human animal thatexpresses antibodies having human variable regions and non-humanconstant regions, which antibodies include light chains that contain ahuman Vλ domain and a non-human Cλ domain, and (2) an engineerednon-human animal that expresses antibodies having human variable regionsand human constant regions, which antibodies include light chains thatcontain human Vλ and Cλ domains. As specifically exemplified herein,expression of such light chains is achieved by insertion of saidplurality of human Vλ and Jλ gene segments into an endogenous Igκ lightchain locus (or allele). In some embodiments, provided non-human animalsare engineered so that expression of endogenous Igλ light chain variableregions is inactivated (e.g., by gene deletion).

In some embodiments, provided non-human animals are engineered so thatexpression of endogenous Igκ light chain variable regions is inactivated(e.g., by replacement or substitution). In some embodiments, providednon-human animals are engineered so that the non-human animals expresshuman Igλ light chain variable regions from an engineered endogenous Igκlight chain locus and human Igκ light chain variable regions from anengineered endogenous Igκ light chain locus. Thus, the presentdisclosure, in at least some embodiments, embraces the development of animproved in vivo system for the production of human antibodies byproviding an engineered non-human animal containing an alternativelyengineered Igκ light chain locus that results in an expressed antibodyrepertoire containing human Vλ domains and non-human or human Cλdomains.

Nucleic Acid Constructs

Typically, a polynucleotide molecule containing human Igλ light chainsequences (e.g., human Vλ and Jλ gene segments) or portion(s) thereoflinked with (e.g., is inserted into) a vector, preferably a DNA vector,in order to replicate the polynucleotide molecule in a host cell.

Human Igλ light chain sequences can be cloned directly from knownsequences or sources (e.g., libraries) or synthesized from germlinesequences designed in silico based on published sequences available fromGenBank or other publically available databases (e.g., IMGT).Alternatively, bacterial artificial chromosome (BAC) libraries canprovide immunoglobulin DNA sequences of interest (e.g., human Vλ and Jλsequences and combinations thereof). BAC libraries can contain an insertsize of 100-150 kb and are capable of harboring inserts as large as 300kb (Shizuya, et al., 1992, Proc. Natl. Acad. Sci., USA 89:8794-8797;Swiatek, et al., 1993, Genes and Development 7:2071-2084; Kim, et al.,1996, Genomics 34 213-218; incorporated herein by reference in theirentireties). For example, a human BAC library harboring average insertsizes of 164-196 kb has been described (Osoegawa, K. et al., 2001,Genome Res. 11(3):483-96; Osoegawa, K. et al., 1998, Genomics 52:1-8,Article No. GE985423, each of which is incorporated herein by referencein its entirety). Human and mouse genomic BAC libraries have beenconstructed and are commercially available (e.g., ThermoFisher). GenomicBAC libraries can also serve as a source of immunoglobulin DNA sequencesas well as transcriptional control regions.

Alternatively, immunoglobulin DNA sequences may be isolated, clonedand/or transferred from yeast artificial chromosomes (YACs). Forexample, the nucleotide sequence of the human Igλ light chain locus hasbeen determined (see, e.g., Dunham, I. et al., 1999, Nature 402:489-95,which is incorporated herein by reference in its entirety). Further,YACs have previously been employed to assemble a human Igλ light chainlocus transgene (see, e.g., Popov, A. V. et al., 1996, Gene 177:195-201;Popov, A. V. et al., 1999, J. Exp. Med. 189(10):1611-19, each of whichis incorporated herein by reference in its entirety). An entire Igλlight chain locus (human or rodent) can be cloned and contained withinseveral YACs. If multiple YACs are employed and contain regions ofoverlapping similarity, they can be recombined within yeast host strainsto produce a single construct representing the entire locus or desiredportions of the locus (e.g., a region to targeted with a targetingvector). YAC arms can be additionally modified with mammalian selectioncassettes by retrofitting to assist in introducing the constructs intoembryonic stems cells or embryos by methods known in the art and/ordescribed herein.

DNA and amino acid sequences of human Igλ light chain gene segments foruse in constructing an engineered Igκ light chain locus as describedherein may be obtained from published databases (e.g., GenBank, IMGT,etc.) and/or published antibody sequences. In some embodiments, nucleicacid constructs containing human Igλ light chain gene segments comprisea J region (i.e., a genomic sequence that includes a plurality of lightchain J gene segments), where the J region comprises coding sequences ofhuman Jλ gene segments with their corresponding 12RSS, where the 12RSShave been positioned among non-coding intergenic DNA typicallyassociated with coding sequences of human Jκ gene segments with theircorresponding 23RSS.

In some embodiments, such a sequence may be referred to as an engineeredlight chain J region. In some certain embodiments, nucleic acidconstructs containing human Igλ light chain gene segments comprise humanVλ and Jλ sequences operably linked to a human or non-human Igλ lightchain constant region (Cλ) gene. In some certain embodiments, nucleicacid constructs containing human Igλ light chain gene segments comprisehuman Vλ and Jλ sequences operably linked to one or more non-human Igκlight chain enhancer regions (or enhancer sequences). In some certainembodiments, nucleic acid constructs containing human Igλ light chaingene segments comprise human Vλ and Jλ sequences operably linked to anon-human or human Cλ region gene and non-human Igκ light chain enhancerregions (or enhancer sequences).

In some embodiments, nucleic acid constructs containing human Vλ and Jλsequences further comprises intergenic DNA that is of human and/ormurine origin. In some embodiments, intergenic DNA is or comprisesnon-coding murine Igκ light chain sequence, non-coding human Igκ lightchain sequence, non-coding murine Igλ light chain sequence, non-codinghuman Igλ light chain sequence, or combinations thereof.

Nucleic acid constructs can be prepared using methods known in the art.For example, a nucleic acid construct can be prepared as part of alarger plasmid. Such preparation allows the cloning and selection of thecorrect constructions in an efficient manner as is known in the art.Nucleic acid constructs containing human Igλ light chain sequences, inwhole or in part, as described herein can be located between restrictionsites on the plasmid so that they can be isolated from the remainingplasmid sequences for incorporation into a desired non-human animal.

Various methods employed in preparation of nucleic acid constructs(e.g., plasmids) and transformation of host organisms are known in theart. For other suitable expression systems for both prokaryotic andeukaryotic cells, as well as general recombinant procedures, seePrinciples of Gene Manipulation: An Introduction to GeneticManipulation, 5th Ed., ed. By Old, R. W. and S. B. Primrose, BlackwellScience, Inc., 1994 and Molecular Cloning: A Laboratory Manual, 2nd Ed.,ed. by Sambrook, J. et al., Cold Spring Harbor Laboratory Press: 1989,each of which is incorporated herein by reference in its entirety.

Targeting Vectors

Targeting vectors can be employed to introduce a nucleic acid constructinto a genomic target locus and comprise a nucleic acid construct andhomology arms that flank said nucleic acid construct; those skilled inthe art will be aware of a variety of options and features generallyapplicable to the design, structure, and/or use of targeting vectors.For example, targeting vectors can be in linear form or in circularform, and they can be single-stranded or double-stranded. Targetingvectors can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).For ease of reference, homology arms are referred to herein as 5′ and 3′(i.e., upstream and downstream) homology arms. This terminology relatesto the relative position of the homology arms to a nucleic acidconstruct within a targeting vector. 5′ and 3′ homology arms correspondto regions within a targeted locus or to a region within anothertargeting vector, which are referred to herein as “5′ target sequence”and “3′ target sequence,” respectively. In some embodiments, homologyarms can also function as a 5′ or a 3′ target sequence.

In some embodiments, methods described herein employ two, three or moretargeting vectors that are capable of recombining with each other. Invarious embodiments, targeting vectors are large targeting vectors(LTVEC) as described elsewhere herein. In such embodiments, first,second, and third targeting vectors each comprise a 5′ and a 3′ homologyarm. The 3′ homology arm of the first targeting vector comprises asequence that overlaps with the 5′ homology arm of the second targetingvector (i.e., overlapping sequences), which allows for homologousrecombination between first and second LTVECs.

In the case of double targeting methods, a 5′ homology arm of a firsttargeting vector and a 3′ homology arm of a second targeting vector canbe similar to corresponding segments within a target genomic locus(i.e., a target sequence), which can promote homologous recombination ofthe first and the second targeting vectors with corresponding genomicsegments and modifies the target genomic locus.

In the case of triple targeting methods, a 3′ homology arm of a secondtargeting vector can comprise a sequence that overlaps with a 5′homology arm of a third targeting vector (i.e., overlapping sequences),which can allow for homologous recombination between the second and thethird LTVEC. The 5′ homology arm of the first targeting vector and the3′ homology arm of the third targeting vector are similar tocorresponding segments within the target genomic locus (i.e., the targetsequence), which can promote homologous recombination of the first andthe third targeting vectors with the corresponding genomic segments andmodifies the target genomic locus.

A homology arm and a target sequence or two homology arms “correspond”or are “corresponding” to one another when the two regions share asufficient level of sequence identity to one another to act assubstrates for a homologous recombination reaction. The sequenceidentity between a given target sequence and the corresponding homologyarm found on a targeting vector (i.e., overlapping sequence) or betweentwo homology arms can be any degree of sequence identity that allows forhomologous recombination to occur. To give but one example, an amount ofsequence identity shared by a homology arm of a targeting vector (or afragment thereof) and a target sequence of another targeting vector or atarget sequence of a target genomic locus (or a fragment thereof) canbe, e.g., but not limited to, at least 50%, 55%, 60%, 65%, 70%, 75%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, such that thesequences undergo homologous recombination.

Moreover, a corresponding region of similarity (e.g., identity) betweena homology arm and a corresponding target sequence can be of any lengththat is sufficient to promote homologous recombination at the targetgenomic locus. For example, a given homology arm and/or correspondingtarget sequence can comprise corresponding regions of similarity thatare, e.g., but not limited to, about 5-10 kb, 5-15 kb, 5-20 kb, 5-25 kb,5-30 kb, 5-35 kb, 5-40 kb, 5-45 kb, 5-50 kb, 5-55 kb, 5-60 kb, 5-65 kb,5-70 kb, 5-75 kb, 5-80 kb, 5-85 kb, 5-90 kb, 5-95 kb, 5-100 kb, 100-200kb, or 200-300 kb in length (such as described elsewhere herein) suchthat a homology arm has sufficient similarity to undergo homologousrecombination with a corresponding target sequence(s) within a targetgenomic locus of the cell or within another targeting vector. In someembodiments, a given homology arm and/or corresponding target sequencecomprise corresponding regions of similarity that are, e.g., but notlimited to, about 10-100 kb, 15-100 kb, 20-100 kb, 25-100 kb, 30-100 kb,35-100 kb, 40-100 kb, 45-100 kb, 50-100 kb, 55-100 kb, 60-100 kb, 65-100kb, 70-100 kb, 75-100 kb, 80-100 kb, 85-100 kb, 90-100 kb, or 95-100 kbin length (such as described elsewhere herein) such that a homology armhas sufficient similarity to undergo homologous recombination with acorresponding target sequence(s) within a target genomic locus of thecell or within another targeting vector.

Overlapping sequences of a 3′ homology arm of a first targeting vectorand a 5′ homology arm of a second targeting vector or of a 3′ homologyarm of a second targeting vector and a 5′ homology arm of a thirdtargeting vector can be of any length that is sufficient to promotehomologous recombination between said targeting vectors. For example, agiven overlapping sequence of a homology arm can comprise correspondingoverlapping regions that are about 1-5 kb, 5-10 kb, 5-15 kb, 5-20 kb,5-25 kb, 5-30 kb, 5-35 kb, 5-40 kb, 5-45 kb, 5-50 kb, 5-55 kb, 5-60 kb,5-65 kb, 5-70 kb, 5-75 kb, 5-80 kb, 5-85 kb, 5-90 kb, 5-95 kb, 5-100 kb,100-200 kb, or 200-300 kb in length such that an overlapping sequence ofa homology arm has sufficient similarity to undergo homologousrecombination with a corresponding overlapping sequence within anothertargeting vector. In some embodiments, a given overlapping sequence of ahomology arm comprises an overlapping region that is about 1-100 kb,5-100 kb, 10-100 kb, 15-100 kb, 20-100 kb, 25-100 kb, 30-100 kb, 35-100kb, 40-100 kb, 45-100 kb, 50-100 kb, 55-100 kb, 60-100 kb, 65-100 kb,70-100 kb, 75-100 kb, 80-100 kb, 85-100 kb, 90-100 kb, or 95-100 kb inlength such that an overlapping sequence of a homology arm hassufficient similarity to undergo homologous recombination with acorresponding overlapping sequence within another targeting vector. Insome embodiments, an overlapping sequence is from 1-5 kb, inclusive. Insome embodiments, an overlapping sequence is from about 1 kb to about 70kb, inclusive. In some embodiments, an overlapping sequence is fromabout 10 kb to about 70 kb, inclusive. In some embodiments, anoverlapping sequence is from about 10 kb to about 50 kb, inclusive. Insome embodiments, an overlapping sequence is at least 10 kb. In someembodiments, an overlapping sequence is at least 20 kb. For example, anoverlapping sequence can be from about 1 kb to about 5 kb, inclusive,from about 5 kb to about 10 kb, inclusive, from about 10 kb to about 15kb, inclusive, from about 15 kb to about 20 kb, inclusive, from about 20kb to about 25 kb, inclusive, from about 25 kb to about 30 kb,inclusive, from about 30 kb to about 35 kb, inclusive, from about 35 kbto about 40 kb, inclusive, from about 40 kb to about 45 kb, inclusive,from about 45 kb to about 50 kb, inclusive, from about 50 kb to about 60kb, inclusive, from about 60 kb to about 70 kb, inclusive, from about 70kb to about 80 kb, inclusive, from about 80 kb to about 90 kb,inclusive, from about 90 kb to about 100 kb, inclusive, from about 100kb to about 120 kb, inclusive, from about 120 kb to about 140 kb,inclusive, from about 140 kb to about 160 kb, inclusive, from about 160kb to about 180 kb, inclusive, from about 180 kb to about 200 kb,inclusive, from about 200 kb to about 220 kb, inclusive, from about 220kb to about 240 kb, inclusive, from about 240 kb to about 260 kb,inclusive, from about 260 kb to about 280 kb, inclusive, or about 280 kbto about 300 kb, inclusive. To give but one example, an overlappingsequence can be from about 20 kb to about 60 kb, inclusive.Alternatively, an overlapping sequence can be at least 1 kb, at least 5kb, at least 10 kb, at least 15 kb, at least 20 kb, at least 25 kb, atleast 30 kb, at least 35 kb, at least 40 kb, at least 45 kb, at least 50kb, at least 60 kb, at least 70 kb, at least 80 kb, at least 90 kb, atleast 100 kb, at least 120 kb, at least 140 kb, at least 160 kb, atleast 180 kb, at least 200 kb, at least 220 kb, at least 240 kb, atleast 260 kb, at least 280 kb, or at least 300 kb. In some embodiments,an overlapping sequence can be at most 400 kb, at most 350 kb, at most300 kb, at most 280 kb, at most 260 kb, at most 240 kb, at most 220 kb,at most 200 kb, at most 180 kb, at most 160 kb, at most 140 kb, at most120 kb, at most 100 kb, at most 90 kb, at most 80 kb, at most 70 kb, atmost 60 kb or at most 50 kb.

Homology arms can, in some embodiments, correspond to a locus that isnative to a cell (e.g., a targeted locus), or alternatively they cancorrespond to a region of a heterologous or exogenous segment of DNAthat was integrated into the genome of the cell, including, for example,transgenes, expression cassettes, or heterologous or exogenous regionsof DNA. In some embodiments, homology arms can, in some embodiments,correspond to a region on a targeting vector in a cell. In someembodiments, homology arms of a targeting vector may correspond to aregion of a yeast artificial chromosome (YAC), a bacterial artificialchromosome (BAC), a human artificial chromosome, or any other engineeredregion contained in an appropriate host cell. Still further, homologyarms of a targeting vector may correspond to or be derived from a regionof a BAC library, a cosmid library, or a P1 phage library. In somecertain embodiments, homology arms of a targeting vector correspond to alocus that is native, heterologous, or exogenous to a prokaryote, ayeast, a bird (e.g., chicken), a non-human mammal, a rodent, a human, arat, a mouse, a hamster a rabbit, a pig, a bovine, a deer, a sheep, agoat, a cat, a dog, a ferret, a primate (e.g., marmoset, rhesus monkey),a domesticated mammal, an agricultural mammal, or any other organism ofinterest. In some embodiments, homology arms correspond to a locus ofthe cell that shows limited susceptibility to targeting using aconventional method or that has shown relatively low levels ofsuccessful integration at a targeted site, and/or significant levels ofoff-target integration, in the absence of a nick or double-strand breakinduced by a nuclease agent (e.g., a Cas protein). In some embodiments,homology arms are designed to include engineered DNA.

In some embodiments, 5′ and 3′ homology arms of a targeting vector(s)correspond to a targeted genome. Alternatively, homology arms correspondto a related genome. For example, a targeted genome is a mouse genome ofa first strain, and targeting arms correspond to a mouse genome of asecond strain, wherein the first strain and the second strain aredifferent. In certain embodiments, homology arms correspond to thegenome of the same animal or are from the genome of the same strain,e.g., the targeted genome is a mouse genome of a first strain, and thetargeting arms correspond to a mouse genome from the same mouse or fromthe same strain.

A homology arm of a targeting vector can be of any length that issufficient to promote a homologous recombination event with acorresponding target sequence, including, for example, 1-5 kb,inclusive, 5-10 kb, inclusive, 5-15 kb, inclusive, 5-20 kb, inclusive,5-25 kb, inclusive, 5-30 kb, inclusive, 5-35 kb, inclusive, 5-40 kb,inclusive, 5-45 kb, inclusive, 5-50 kb, inclusive, 5-55 kb, inclusive,5-60 kb, inclusive, 5-65 kb, inclusive, 5-70 kb, inclusive, 5-75 kb,inclusive, 5-80 kb, inclusive, 5-85 kb, inclusive, 5-90 kb, inclusive,5-95 kb, inclusive, 5-100 kb, inclusive, 100-200 kb, inclusive, or200-300 kb, inclusive, in length. In some embodiments, a homology arm ofa targeting vector has a length that is sufficient to promote ahomologous recombination event with a corresponding target sequence thatis 1-100 kb, inclusive, 5-100 kb, inclusive, 10-100 kb, inclusive,15-100 kb, inclusive, 20-100 kb, inclusive, 25-100 kb, inclusive, 30-100kb, inclusive, 35-100 kb, inclusive, 40-100 kb, inclusive, 45-100 kb,inclusive, 50-100 kb, inclusive, 55-100 kb, inclusive, 60-100 kb,inclusive, 65-100 kb, inclusive, 70-100 kb, inclusive, 75-100 kb,inclusive, 80-100 kb, inclusive, 85-100 kb, inclusive, 90-100 kb,inclusive, or 95-100 kb, inclusive, in length. As described herein,large targeting vectors can employ targeting arms of greater length.

Nuclease agents (e.g., CRISPR/Cas systems) can be employed incombination with targeting vectors to facilitate the modification of atarget locus (e.g., modification of an Igκ light chain locus, ormodification of a previously modified or engineered Igκ light chainlocus). Such nuclease agents may promote homologous recombinationbetween a targeting vector and a target locus. When nuclease agents areemployed in combination with a targeting vector, the targeting vectorcan comprise 5′ and 3′ homology arms corresponding to 5′ and 3′ targetsequences located in sufficient proximity to a nuclease cleavage site soas to promote the occurrence of a homologous recombination event betweentarget sequences and homology arms upon a nick or double-strand break atthe nuclease cleavage site. The term “nuclease cleavage site” includes aDNA sequence at which a nick or double-strand break is created by anuclease agent (e.g., a Cas9 cleavage site). Target sequences within atargeted locus that correspond to 5′ and 3′ homology arms of a targetingvector are “located in sufficient proximity” to a nuclease cleavage siteif the distance is such as to promote the occurrence of a homologousrecombination event between 5′ and 3′ target sequences and homology armsupon a nick or double-strand break at the recognition site. Thus, incertain embodiments, target sequences corresponding to 5′ and/or 3′homology arms of a targeting vector are within at least one nucleotideof a given recognition site or are within at least 10 nucleotides toabout 14 kb of a given recognition site. In some embodiments, a nucleasecleavage site is immediately adjacent to at least one or both of thetarget sequences.

The spatial relationship of target sequences that correspond to homologyarms of a targeting vector and a nuclease cleavage site can vary. Forexample, target sequences can be located 5′ to a nuclease cleavage site,target sequences can be located 3′ to a recognition site, or targetsequences can flank a nuclease cleavage site.

Combined use of a targeting vector (including, for example, a largetargeting vector) with a nuclease agent can result in an increasedtargeting efficiency compared to use of a targeting vector alone. Forexample, when a targeting vector is used in conjunction with a nucleaseagent, targeting efficiency of a targeting vector can be increased by atleast two-fold, at least three-fold, at least four-fold, at leastfive-fold, at least six-fold, at least seven-fold, at least eight-fold,at least nine-fold, at least ten-fold or within a range formed fromthese integers, such as 2-10-fold when compared to use of a targetingvector alone.

Some targeting vectors are “large targeting vectors” or “LTVECs,” whichincludes targeting vectors that comprise homology arms that correspondto and are derived from nucleic acid sequences larger than thosetypically used by other approaches intended to perform homologousrecombination in cells. A LTVEC can be, for example, at least 10 kb inlength, or the sum total of a 5′ homology arm and a 3′ homology arm canbe, for example, at least 10 kb. LTVECs also include targeting vectorscomprising nucleic acid constructs larger than those typically used byother approaches intended to perform homologous recombination in cells.For example, LTVECs make possible the modification of large loci thatcannot be accommodated by traditional plasmid-based targeting vectorsbecause of their size limitations. For example, a targeted locus can be(i.e., 5′ and 3′ homology arms can correspond to) a locus of a cell thatis not targetable using a conventional method or that can be targetedonly incorrectly or only with significantly low efficiency in theabsence of a nick or double-strand break induced by a nuclease agent(e.g., a Cas protein).

In some embodiments, methods described herein employ two or three LTVECsthat are capable of recombining with each other and with a targetgenomic locus in a three-way or a four-way recombination event. Suchmethods make possible the modification of large loci that cannot beachieved using a single LTVEC.

Examples of LTVECs include vectors derived from a bacterial artificialchromosome (BAC), a human artificial chromosome, or a yeast artificialchromosome (YAC). LTVECs can be in linear form or in circular form.Examples of LTVECs and methods for making them are described, e.g., inU.S. Pat. Nos. 6,586,251, 6,596,541 and 7,105,348; and InternationalPatent Application Publication No. WO 2002/036789, each of which isincorporated herein by reference in their entireties.

Provided Non-Human Animals, Cells and Tissues

Non-human animals are provided that express (e.g., whose B cellsexpress) antibodies that contain light chains that include a human Vλdomain resulting from integration of genetic material that correspondsto at least a portion of a human Igλ light chain locus (i.e., at least aportion of human Vλ and Jλ gene segments), and which encodes a human Vλdomain (i.e., a rearranged human Vλ-Jλ sequence), in the place ofcorresponding non-human Igκ light chain variable region sequences in thegermline genome of the non-human animal. Suitable examples describedherein include, but are not limited to, rodents, in particular, mice.

The present disclosure provides improved in vivo systems for identifyingand developing new antibodies, antibody components (e.g.,antigen-binding portions and/or compositions or formats that includethem), and/or antibody-based therapeutics that can be used, for example,in the treatment of a variety of diseases that affect humans. Further,the present disclosure also encompasses the recognition that non-humananimals (e.g., rodents) having engineered immunoglobulin loci, such asengineered immunoglobulin (Ig) kappa (κ) light chain loci and/orotherwise expressing, producing or containing antibody repertoirescharacterized by light chains having human V lambda (λ) regions areuseful. For example, in some embodiments, such non-human animals may beused for exploiting the diversity of human Vλ sequences in theidentification and development of new antibody-based therapeutics. Insome embodiments, non-human animals described herein provide improved invivo systems for development of antibodies and/or antibody-basedtherapeutics for administration to humans. In some embodiments,non-human animals described herein provide improved in vivo systems fordevelopment of antibodies and/or antibody-based therapeutics thatcontain human Vλ domains characterized by improved performance (e.g.,expression and/or representation in an antigen-specific antibodyrepertoire) as compared to antibodies and/or antibody-based therapeuticsobtained from existing in vivo systems that contain human Vλ regionsequences.

The present disclosure provides, among other things, a non-human animalhaving an Igκ light chain locus that contains an engineeredimmunoglobulin light chain variable region and an engineeredimmunoglobulin light chain constant region gene. As described herein,provided non-human animals, contain in their germline genome animmunoglobulin κ light chain locus comprising an engineeredimmunoglobulin κ light chain variable region characterized by thepresence of one or more human Vλ gene segments and one or more human Jλgene segments, which one or more human Vλ and one or more human Jλ genesegments are operably linked to an immunoglobulin λ light chain constantregion (Cλ) gene, which immunoglobulin λ light chain constant region(Cλ) gene is positioned in the place of a non-human immunoglobulin κlight chain constant region (Cκ) gene at the endogenous immunoglobulin κlocus of the non-human animal. In some embodiments, provided non-humananimals comprise an Igκ light chain locus that contains intergenic DNAthat is immunoglobulin λ light chain and/or immunoglobulin κ light chainin origin, and combinations thereof.

In many embodiments, an engineered immunoglobulin κ light chain variableregion further comprises an immunoglobulin κ light chain sequencepositioned or inserted between said one or more human Vλ gene segmentsand one or more human Jλ gene segments. In some embodiments, saidimmunoglobulin κ light chain sequence positioned or inserted betweensaid one or more human Vλ gene segments and one or more human Jλ genesegments is or comprises a murine (e.g., rat or mouse) sequence. In someembodiments, said immunoglobulin κ light chain sequence positioned orinserted between said one or more human Vλ gene segments and one or morehuman Jλ gene segments is or comprises a human sequence. For example, insome embodiments, a human immunoglobulin κ light chain sequence is orcomprises a genomic sequence that naturally appears between a humanVκ4-1 gene segment and a human Jκ1 gene segment of a humanimmunoglobulin κ light chain locus.

In some embodiments, provided non-human animals comprise at least 5, atleast 6, at least 7, at least 8, at least 9, at least 10, at least 11,at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, at least 19, at least 20, at least 21, at least22, at least 23, at least 24 or at least 25 functional human Vλ genesegments. In some embodiments, provided non-human animals comprise 5 to25, 5 to 24, 5 to 23, 5 to 22, 5 to 21, 5 to 20, 5 to 19, 5 to 18, 5 to17, 5 to 16, 5 to 15, 5 to 14, 5 to 13, 5 to 12, 5 to 11, 5 to 10, 5 to9, 5 to 8, 5 to 7, or 5 to 6 functional human Vλ gene segments. In someembodiments, provided non-human animals comprise 6 to 25, 7 to 25, 8 to25, 9 to 25, 10 to 25, 11 to 25, 12 to 25, 13 to 25, 14 to 25, 15 to 25,16 to 25, 17 to 25, 18 to 25, 19 to 25, 20 to 25, 21 to 25, 22 to 25, 23to 25 or 24 to 25 functional human Vλ gene segments. In someembodiments, provided non-human animals comprise 6 to 24, 7 to 23, 8 to22, 9 to 21, 10 to 20, 11 to 19, 12 to 18, 13 to 17, 14 to 16, or 15 to16 functional human Vλ gene segments. In some embodiments, providednon-human animals comprise 6 to 24, 7 to 23, 8 to 22, 9 to 21, 10 to 20,11 to 19, 12 to 18, 13 to 17, or 14 to 16 functional human Vλ genesegments.

In some embodiments, provided non-human animals comprise 10 to 70, 10 to65, 10 to 60, 10 to 55, 10 to 50, 10 to 45, 10 to 40, 10 to 35, 10 to30, 10 to 25, 10 to 20, or 10 to 15 total human Vλ gene segments. Insome embodiments, provided non-human animals comprise 15 to 70, 20 to70, 25 to 70, 30 to 70, 35 to 70, 40 to 70, 45 to 70, 50 to 70, 55 to70, 60 to 70, or 65 to 70 total human Vλ gene segments. In someembodiments, provided non-human animals comprise 15 to 65, 20 to 60, 25to 55, 20 to 50, 25 to 45, 30 to 40, 30 to 35, or 35 to 40 total humanVλ gene segments.

In some embodiments, provided non-human animals contain human Vλ and/orJλ gene segments in natural or germline configuration (e.g., a DNAsequence containing a plurality of human Vλ and/or Jλ gene segmentcoding sequences interspersed with non-coding human immunoglobulin λlight chain sequence light chain sequence). In some embodiments,provided non-human animals contain human Vλ and/or Jλ gene segments inconfiguration that departs or deviates from a natural or germlineconfiguration (e.g., a DNA sequence containing a plurality of human Vλand/or Jλ gene segment coding sequences interspersed with non-codingimmunoglobulin κ light chain sequence (e.g., human or murine]). In someembodiments, provided non-human animals contain human Vλ and/or Jλ genesegments in a configuration that does not naturally appear in a humanimmunoglobulin λ light chain locus of the germline genome of a humancell.

In some embodiments, provided non-human animals contain a DNA sequenceat an endogenous non-human Igκ light chain locus that includes aplurality of human Vλ and Jλ coding sequences interspersed (orjuxtaposed, associated, etc.) with non-coding human immunoglobulin lightchain sequence (e.g., κ, λ and combinations thereof). In someembodiments, provided non-human animals contain a DNA sequence at anendogenous non-human Igλ light chain locus that includes a plurality ofhuman Vλ and Jλ coding sequences interspersed with non-coding non-human(e.g., murine) immunoglobulin λ light chain sequence.

In some embodiments, provided non-human animals are characterized byexpression of antibodies from endogenous immunoglobulin κ light chainloci in the germline genome of said non-human animals, which antibodiescontain (1) human Vλ domains and (2) non-human or human Cλ domains. Insome embodiments, provided non-human animals are characterized by animproved usage of human Vλ regions from engineered immunoglobulin κlight chain loci (e.g., but not limited to, about 2-fold) as compared toone or more reference engineered non-human animals.

In some embodiments, a non-human animal, non-human cell or non-humantissue is provided whose germline genome comprises an endogenousimmunoglobulin κ light chain locus comprising: (a) one or more human Vλgene segments, (b) one or more human Jλ gene segments, and (c) a Cλgene, wherein (a) and (b) are operably linked to (c), and wherein therodent lacks a rodent Cκ gene at the endogenous immunoglobulin κ lightchain locus.

In some embodiments, a non-human animal, non-human cell or non-humantissue is provided whose germline genome comprises an endogenousimmunoglobulin κ light chain locus comprising insertion of one or morehuman Vλ gene segments, one or more human Jλ gene segments and a Cλgene, which human Vλ and Jλ gene segments are operably linked to said Cλgene, and which Cλ gene is inserted in the place of a non-human Cκ geneat the endogenous immunoglobulin κ light chain locus. In manyembodiments of a non-human animal, non-human cell or non-human tissue, aCλ gene inserted in the place of a non-human Cκ gene at an endogenousimmunoglobulin κ light chain locus is a non-human or human Cλ gene. Insome embodiments, a non-human Cλ gene is or comprises a mammalian Cλgene selected from the group consisting of a primate, goat, sheep, pig,dog, cow, or rodent Cλ gene.

In some embodiments, a non-human Cλ gene is or comprises a rodent Cλgene.

In some embodiments, a rodent Cλ gene is or comprises a mouse Cλ gene.In some embodiments, a mouse Cλ gene comprises a sequence that is atleast 80%, at least 85%, at least 90%, at least 95%, or at least 98%identical to a mouse Cλ gene selected from the group consisting of amouse Cλ1, mouse Cλ2 and a mouse Cλ3. In some embodiments, a mouse Cλgene comprises a sequence that is substantially identical or identicalto a mouse Cλ gene selected from the group consisting of a mouse Cλ1,mouse Cλ2 and a mouse Cλ3. In some embodiments, a mouse Cλ1 gene is orcomprises SEQ ID NO: 1. In some certain embodiments, a mouse Cλ2 gene isor comprises SEQ ID NO:3. In some certain embodiments, a mouse Cλ3 geneis or comprises SEQ ID NO:5. In some certain embodiments, a mouse Cλgene comprises a sequence that is identical to a mouse Cλ1 gene.

In some embodiments, a mouse Cλ gene comprises a sequence that is 80% to100%, 85% to 100%, 90% to 100%, 95% to 100%, or 98% to 100% identical toa mouse Cλ gene selected from the group consisting of a mouse Cλ1, mouseCλ2 and a mouse Cλ3. In some embodiments, a mouse Cλ gene comprises asequence that is 80% to 98%, 80% to 95%, 80% to 90%, or 80% to 85%identical to a mouse Cλ gene selected from the group consisting of amouse Cλ1, mouse Cλ2 and a mouse Cλ3. In some embodiments, a mouse Cλgene comprises a sequence that is 85% to 98%, 90% to 95%, or 88% to 93%identical to a mouse Cλ gene selected from the group consisting of amouse Cλ1, mouse Cλ2 and a mouse Cλ3.

In some embodiments, a rodent Cλ gene is or comprises a rat Cλ gene. Insome embodiments, a rat Cλ gene comprises a sequence that is at least80%, at least 85%, at least 90%, at least 95%, or at least 98% identicalto a rat Cλ gene selected from the group consisting of a rat Cλ1, ratCλ2, rat Cλ3 and a rat Cλ4 gene. In some embodiments, a rat Cλ genecomprises a sequence that is substantially identical or identical to arat Cλ gene selected from the group consisting of a rat Cλ1, rat Cλ2,rat Cλ3 and a rat Cλ4 gene. In some certain embodiments, a rat Cλ1 geneis or comprises SEQ ID NO:7. In some certain embodiments, a rat Cλ2 geneis or comprises SEQ ID NO:9. In some certain embodiments, a rat Cλ3 geneis or comprises SEQ ID NO: 11. In some certain embodiments, a rat Cλ4gene is or comprises SEQ ID NO:13.

In some embodiments, a rat Cλ gene comprises a sequence that is 80% to100%, 85% to 100%, 90% to 100%, 95% to 100%, or 98% to 100% identical toa rat Cλ gene selected from the group consisting of a rat Cλ1, rat Cλ2,rat Cλ3 and a rat Cλ4 gene. In some embodiments, a rat Cλ gene comprisesa sequence that is 80% to 98%, 80% to 95%, 80% to 90%, or 80% to 85%identical to a rat Cλ gene selected from the group consisting of a ratCλ1, rat Cλ2, rat Cλ3 and a rat Cλ4 gene. In some embodiments, a rat Cλgene comprises a sequence that is 85% to 98%, 90% to 95%, or 88% to 93%,identical to a rat Cλ gene selected from the group consisting of a ratCλ1, rat Cλ2, rat Cλ3 and a rat Cλ4 gene.

In some embodiments, a human Cλ gene comprises a sequence that is atleast 80%, at least 85%, at least 90%, at least 95%, or at least 98%identical to a human Cλ gene selected from the group consisting of ahuman Cλ1, human Cλ2, human Cλ3, human Cλ6 and a human Cλ7 gene. In someembodiments, a human Cλ gene comprises a sequence that is substantiallyidentical or identical to a human Cλ gene selected from the groupconsisting of a human Cλ, human Cλ2, human Cλ3, human Cλ6 and a humanCλ7 gene. In some embodiments, a human Cλ gene comprises a sequence thatis identical to a human Cλ gene selected from the group consisting of ahuman Cλ1, human Cλ2, human Cλ3, human Cλ6 and a human Cλ7 gene. In somecertain embodiments, a human Cλ1 gene is or comprises SEQ ID NO:15. Insome certain embodiments, a human Cλ2 gene is or comprises SEQ ID NO:17.In some certain embodiments, a human Cλ3 gene is or comprises SEQ ID NO:19. In some certain embodiments, a human Cλ6 gene is or comprises SEQ IDNO:21. In some certain embodiments, a human Cλ7 gene is or comprises SEQID NO:23. In some certain embodiments, a human Cλ gene is or comprises ahuman Cλ2 gene.

In some embodiments, a human Cλ gene comprises a sequence that is 80% to100%, 85% to 100%, 90% to 100%, 95% to 100%, or 98% to 100% identical toa human Cλ gene selected from the group consisting of a human Cλ1, humanCλ2, human Cλ3, human Cλ6 and a human Cλ7 gene. In some embodiments, ahuman Cλ gene comprises a sequence that is 80% to 98%, 80% to 95%, 80%to 90%, or 80% to 85% identical to a human Cλ gene selected from thegroup consisting of a human Cλ1, human Cλ2, human Cλ3, human Cλ6 and ahuman Cλ7 gene. In some embodiments, a human Cλ gene comprises asequence that is 85% to 98%, 90% to 95%, or 88% to 93%, identical to ahuman Cλ gene selected from the group consisting of a human Cλ1, humanCλ2, human Cλ3, human Cλ6 and a human Cλ7 gene.

In some embodiments of a provided non-human animal, non-human cell ornon-human tissue, insertion of one or more human Vλ gene segments andone or more human Jλ gene segments replace non-human Vκ and Jκ genesegments at the endogenous immunoglobulin κ light chain locus. In someembodiments, insertion includes human non-coding DNA that naturallyappears between human Vλ and Jλ gene segments, and combinations thereof.In some embodiments of a provided non-human animal, non-human cell ornon-human tissue, insertion of one or more human Vλ gene segments andone or more human Jλ gene segments are in place of or replace non-humanVκ and Jκ gene segments at the endogenous immunoglobulin κ light chainlocus. In some embodiments of a provided non-human animal, non-humancell or non-human tissue, an immunoglobulin κ light chain locuscomprises insertion of at least 24, at least 34, at least 52, at least61, or at least 70 human Vλ gene segments and at least 1, at least 2, atleast 3, at least 4 or at least 5 human Jλ gene segments. In somecertain embodiments of a provided non-human animal, non-human cell ornon-human tissue, an immunoglobulin κ light chain locus comprisesinsertion of 39 human Vλ gene segments and at least 5 human Jλ genesegments. In some embodiments of a provided non-human animal, non-humancell or non-human tissue, an immunoglobulin κ light chain locuscomprises insertion of human Vλ4-69, Vλ8-61, Vλ4-60, Vλ6-57, Vλ10-54,Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40,Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19,Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, V,3-1 orany combination thereof, and human J Jλ1, Jλ2, Jλ3, Jλ6, Jλ7, or anycombination thereof. In some embodiments, insertion includes humannon-coding DNA that naturally appears adjacent to a human Vλ4-69,Vλ8-61, Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46,Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25,Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10,Vλ3-9, Vλ2-8, Vλ4-3, or Vλ3-1 in an endogenous human λ light chainlocus, and human non-coding DNA (in whole or in part) that naturallyappears adjacent to a human Jλ1, Jλ2, Jλ3, Jλ6 or Jλ7 in an endogenoushuman λ light chain locus. In some certain embodiments, insertionincludes human non-coding DNA that naturally appears adjacent to a humanVλ4-69, Vλ8-61, Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47,Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27,Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11,Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, or Vλ3-1 in an endogenous human λ lightchain locus, and human non-coding DNA that naturally appears adjacent toa human Jκ 1, Jκ2, Jκ3, Jκ4, or Jκ5 in an endogenous human κ light chainlocus. In some certain embodiments, insertion of human Vλ4-69, Vλ8-61,Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45,Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23,Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9,Vλ2-8, Vλ4-3, Vλ3-1 or any combination thereof includes human non-codingDNA that naturally appears adjacent to a human Vλ4-69, Vλ8-61, Vλ4-60,Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44,Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22,Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8,Vλ4-3, or Vλ3-1 in an endogenous human λ light chain locus, and theinsertion of human Jλ1, Jλ2, Jλ3, Jλ6, Jλ7, or any combination thereofincludes human non-coding DNA (in whole or in part) that naturallyappears adjacent to a human Jλ1, Jλ2, Jλ3, Jλ6, Jλ7 in an endogenoushuman λ light chain locus. In some certain embodiments, the insertion ofhuman Vλ4-69, Vλ8-61, Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49,Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36,Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14, Vλ3-12,Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, Vλ3-1 or any combination thereofincludes human non-coding DNA that naturally appears adjacent to a humanVλ4-69, Vλ8-61, Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47,Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27,Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11,Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, or Vλ3-1 in an endogenous human λ lightchain locus, and the insertion of human Jλ1, Jλ2, Jλ3, Jλ6, Jλ7, or anycombination thereof includes human non-coding DNA (in whole or in part)that naturally appears adjacent to a human Jκ1, Jκ2, Jκ3, Jκ4, or Jκ5 inan endogenous human κ light chain locus.

In some embodiments of a provided non-human animal, non-human cell ornon-human tissue, an immunoglobulin κ light chain locus as describedherein further comprises a human immunoglobulin κ light chain sequencebetween the one or more human Vλ gene segments, the one or more human Jλgene segments, the one or more human Vλ gene segments and the one ormore human Jλ gene segments, and combinations thereof. In someembodiments, a human immunoglobulin κ light chain sequence as describedherein is or comprises a genomic sequence that naturally appears betweena human Vκ4-1 gene segment and a human Jκ1 gene segment of a humanimmunoglobulin κ light chain locus.

In some embodiments of a provided non-human animal, non-human cell ornon-human tissue, the germline genome of said non-human animal,non-human cell or non-human tissue further comprises an endogenousimmunoglobulin heavy chain locus comprising insertion of one or morehuman V_(H) gene segments, one or more human D_(H) gene segments and oneor more human J_(H) gene segments, which human V_(H), D_(H) and J_(H)gene segments are operably linked to a non-human immunoglobulin heavychain constant region at the endogenous immunoglobulin heavy chain locus(see, e.g., U.S. Pat. Nos. 8,502,018, 8,642,835, 8,697,940 and8,791,323, each of which is incorporated herein by reference in itsentirety).

In some embodiments, insertion of one or more human V_(H) gene segments,one or more human D_(H) gene segments and one or more human J_(H) genesegments are in place of or replace, in whole or in part, non-humanV_(H), D_(H) and J_(H) gene segments (e.g., positionally replace orsubstitute coding sequences of non-human V_(H), D_(H) and J_(H) genesegments with coding sequences of human V_(H), D_(H) and J_(H) genesegments). In some certain embodiments, insertion includes humannon-coding DNA that naturally appears between human V_(H), D_(H) andJ_(H) gene segments, and combinations thereof. In some embodiments, anon-human immunoglobulin heavy chain constant region is or comprises anendogenous non-human immunoglobulin heavy chain constant region. In manyembodiments, a non-human immunoglobulin heavy chain constant region(e.g., endogenous) includes one or more non-human immunoglobulin heavychain constant region genes or gene segments (e.g., IgM, IgD, IgG, IgE,IgA, etc.). In some certain embodiments, an immunoglobulin heavy chainlocus as described herein comprises insertion of the human V_(H) genesegments V_(H)3-74, V_(H)3-73, V_(H)3-72, V_(H)2-70, V_(H)1-69,V_(H)3-66, V_(H)3-64, V_(H)4-61, V_(H)4-59, V_(H)1-58, V_(H)3-53,V_(H)5-51, V_(H)3-49, V_(H)3-48, V_(H)1-46, V_(H)1-45, V_(H)3-43,V_(H)4-39, V_(H)4-34, V_(H)3-33, V_(H)4-31, V_(H)3-30, V_(H)4-28,V_(H)2-26, V_(H)1-24, V_(H)3-23, V_(H)3-21, V_(H)3-20, V_(H)1-18,V_(H)3-15, V_(H)3-13, V_(H)3-11, V_(H)3-9, V_(H)1-8, V_(H)3-7, V_(H)2-5,V_(H)7-4-1, V_(H)4-4, V_(H)1-3, V_(H)1-2, V_(H)6-1, or any combinationthereof, the human D_(H) gene segments D_(H)1-1, D_(H)2-2, D_(H)3-3,D_(H)4-4, D_(H)5-5, D_(H)6-6, D_(H)1-7, D_(H)2-8, D_(H)3-9, D_(H)3-10,D_(H)5-12, D_(H)6-13, D_(H)2-15, D_(H)3-16, D_(H)4-17, D_(H)6-19,D_(H)1-20, D_(H)2-21, D_(H)3-22, D_(H)6-25, D_(H)1-26, D_(H)7-27, or anycombination thereof, and the human J_(H) gene segments J_(H)1, J_(H)2,J_(H)3, J_(H)4, J_(H)5, J_(H)6, or any combination thereof. In somecertain embodiments, insertion includes human non-coding DNA thatnaturally appears adjacent to a human V_(H)3-74, V_(H)3-73, V_(H)3-72,V_(H)2-70, V_(H)1-69, V_(H)3-66, V_(H)3-64, V_(H)4-61, V_(H)4-59,V_(H)1-58, V_(H)3-53, V_(H)5-51, V_(H)3-49, V_(H)3-48, V_(H)1-46,V_(H)1-45, V_(H)3-43, V_(H)4-39, V_(H)4-34, V_(H)3-33, V_(H)4-31,V_(H)3-30, V_(H)4-28, V_(H)2-26, V_(H)1-24, V_(H)3-23, V_(H)3-21,V_(H)3-20, V_(H)1-18, V_(H)3-15, V_(H)3-13, V_(H)3-11, V_(H)3-9,V_(H)1-8, V_(H)3-7, V_(H)2-5, V_(H)7-4-1, V_(H)4-4, V_(H)1-3, V_(H)1-2,or V_(H)6-1 in an endogenous heavy chain locus, human non-coding DNAthat naturally appears adjacent to a human D_(H)1-1, D_(H)2-2, D_(H)3-3,D_(H)4-4, D_(H)5-5, D_(H)6-6, D_(H)1-7, D_(H)2-8, D_(H)3-9, D_(H)3-10,D_(H)5-12, D_(H)6-13, D_(H)2-15, D_(H)3-16, D_(H)4-17, D_(H)6-19,D_(H)1-20, D_(H)2-21, D_(H)3-22, D_(H)6-25, D_(H)1-26, or D_(H)7-27, andhuman non-coding DNA that naturally appears adjacent to a human J_(H)1,J_(H)2, J_(H)3, J_(H)4, J_(H)5, or J_(H) in an endogenous heavy chainlocus.

In some embodiments, a non-human animal described herein includes anAdam6 gene in its genome (e.g., its germline genome), which encodes anADAM6 polypeptide, functional ortholog, functional homolog, orfunctional fragment thereof (see, e.g., U.S. Pat. Nos. 8,642,835 and8,697,940, each of which is incorporated herein by reference in itsentirety). In some embodiments, an ADAM6 polypeptide, functionalortholog, functional homolog, or functional fragment thereof isexpressed from an Adam6 gene. In some embodiments, an Adam6 gene is doesnot originate from the non-human animal that includes an Adam6 gene(e.g., a mouse that includes a rat Adam6 gene or a mouse Adam6 geneobtained from another strain of mouse). In some embodiments, a non-humananimal described herein includes an ectopic Adam6 gene. An “ectopic”Adam6 gene, as used herein, refers to an Adam6 gene that is in adifferent context than the Adam6 gene appears in a wild-type non-humananimal. For example, the Adam6 gene could be located on a differentchromosome, located at a different locus, or positioned adjacent todifferent sequences. An exemplary ectopic Adam6 gene is a mouse Adam6gene located within human immunoglobulin sequences (e.g., human heavychain variable region gene segments). In some embodiments, a non-humananimal described herein includes an inserted or integrated Adam6 gene.

In some embodiments, a non-human animal described herein includes aninsertion of one or more nucleotide sequences encoding one or morenon-human Adam6 polypeptides, functional orthologs, functional homologs,or functional fragments thereof in its genome (e.g., its germlinegenome).

In some embodiments, a non-human animal described herein includes one ormore nucleotide sequences encoding one or more non-human ADAM6polypeptides, functional orthologs, functional homologs, or functionalfragments thereof in its genome (e.g., its germline genome). In someembodiments, a non-human animal described herein includes a mouse Adam6agene and/or a mouse Adam6b gene in its genome (e.g. its germlinegenome). In some embodiments, a non-human animal described hereinincludes one or more nucleotide sequences a mouse ADAM6a, functionalortholog, functional homolog, or functional fragment thereof, and/or amouse ADAM6b, functional ortholog, functional homolog, or functionalfragment thereof.

In some embodiments, one or more nucleotide sequences encoding one ormore non-human ADAM6 polypeptides, functional orthologs, functionalhomologs, or functional fragments thereof are inserted and/or arelocated on the same chromosome as the endogenous immunoglobulin heavychain locus. In some embodiments, one or more nucleotide sequencesencoding one or more non-human ADAM6 polypeptides, functional orthologs,functional homologs, or functional fragments thereof are inserted and/orare located in a position so that the one or more nucleotide sequencesencoding one or more non-human ADAM6 polypeptides, functional orthologs,functional homologs, or functional fragments thereof are contiguous withhuman immunoglobulin heavy chain variable region gene segments. In someembodiments, one or more nucleotide sequences encoding one or morenon-human ADAM6 polypeptides, functional orthologs, functional homologs,or functional fragments thereof are inserted and/or are located in aposition so that the one or more nucleotide sequences encoding one ormore non-human ADAM6 polypeptides, functional orthologs, functionalhomologs, or functional fragments thereof are adjacent to humanimmunoglobulin heavy chain variable region gene segments. In someembodiments, one or more nucleotide sequences encoding one or morenon-human ADAM6 polypeptides, functional orthologs, functional homologs,or functional fragments thereof are inserted and/or are located in aposition so that the one or more nucleotide sequences encoding one ormore non-human ADAM6 polypeptides, functional orthologs, functionalhomologs, or functional fragments thereof are located in between humanimmunoglobulin heavy chain variable region gene segments. In someembodiments, one or more nucleotide sequences encoding one or morenon-human ADAM6 polypeptides, functional orthologs, functional homologs,or functional fragments thereof are inserted and/or are located betweena first and a second human V_(H) gene segment. In some embodiments, afirst human V_(H) gene segment is human V_(H)1-2 and a second humanV_(H) gene segment is human V_(H)6-1. In some embodiments, one or morenucleotide sequences encoding one or more non-human ADAM6 polypeptides,functional orthologs, functional homologs, or functional fragmentsthereof are inserted and/or are located in the place of a human Adam6pseudogene. In some embodiments, one or more nucleotide sequencesencoding one or more non-human ADAM6 polypeptides, functional orthologs,functional homologs, or functional fragments thereof are insertedbetween a human V_(H) gene segment and a human D_(H) gene segment.

In some embodiments, a non-human animal described herein includes anAdam6 gene that restores or enhances ADAM6 activity. In someembodiments, the Adam6 gene restores ADAM6 activity to the level of acomparable non-human animal that includes a functional, endogenous Adam6gene. In some embodiments, the Adam6 gene enhances ADAM6 activity to alevel that is at least 2 times, at least 3 times, at least 4 times, atleast 5 times, at least 6 times, at least 7 times, at least 8 times, atleast 9 times, or at least 10 times the ADAM6 activity of a comparablenon-human animal that does not include a functional Adam6 gene.

In some embodiments, a non-human animal described herein includes anAdam6 gene that restores or enhances fertility in a male non-humananimal. In some embodiments, the Adam6 gene restores fertility in a malenon-human animal to a level of a comparable non-human animal thatincludes a functional, endogenous Adam6 gene. In some embodiments, theAdam6 gene restores fertility in a male non-human animal so that thenumber of pups produced by mating the male non-human animal is at least70%, at least 80%, at least 90%, at least 95% the number of pupsproduced from a comparable mating of a comparable, male non-human animalthat does not include a functional Adam6 gene. In some embodiments, theAdam6 gene enhances fertility in a male non-human animal so that numberof pups produced by the mating of the male non-human animal include atleast 2 times, at least 3 times, at least 4 times, at least 5 times, atleast 6 times, at least 7 times, at least 8 times, at least 9 times, orat least 10 times the number of pups produced from a comparable matingof a comparable, male non-human animal that does not include afunctional Adam6 gene.

In some embodiments, a non-human immunoglobulin heavy chain locus asdescribed herein lacks at least one endogenous non-human Adam6 gene. Insome embodiments, the lack of the at least one endogenous non-humanAdam6 gene reduces ADAM6 activity and/or fertility in a male mouse thatlacks an endogenous non-human Adam6 gene. In some embodiments, anon-human immunoglobulin heavy chain locus as described herein includesa disruption of at least one endogenous non-human Adam6 gene. In someembodiments, the disruption of at least one endogenous non-human Adam6gene reduces ADAM6 activity and/or fertility in a male mouse that lacksan endogenous non-human Adam6 gene.

In some embodiments of a non-human animal, non-human cell or non-humantissue, the non-human animal, non-human cell or non-human tissue ishomozygous or heterozygous for an endogenous immunoglobulin heavy chainlocus as described herein.

In some embodiments of a non-human animal, non-human cell or non-humantissue, the non-human animal, non-human cell or non-human tissue ishomozygous or heterozygous for an endogenous immunoglobulin κ lightchain locus as described herein.

In some embodiments of a provided non-human animal, non-human cell ornon-human tissue, the endogenous immunoglobulin λ light chain locus isdeleted in whole or in part. In some embodiments of a provided non-humananimal, non-human cell or non-human tissue, the endogenousimmunoglobulin λ light chain locus is functionally silenced or otherwisenon-functional (e.g., by gene targeting). In some certain embodiments ofa provided non-human animal, non-human cell or non-human tissue, thenon-human animal, non-human cell or non-human tissue is homozygous orheterozygous for a functionally silenced or otherwise non-functionalendogenous immunoglobulin λ light chain locus as described herein.

In some embodiments, a non-human animal, non-human cell or non-humantissue as described herein does not detectably express endogenousimmunoglobulin λ light chains, endogenous immunoglobulin κ light chains,or endogenous immunoglobulin λ light chains and endogenousimmunoglobulin κ light chains.

In some embodiments, a non-human animal, non-human cell or non-humantissue as described herein has a genome further comprising a nucleicacid sequence encoding an exogenous terminal deoxynucleotidyltransferase(TdT) operably linked to a transcriptional control element.

In some embodiments, a transcriptional control element includes a RAG1transcriptional control element, a RAG2 transcriptional control element,an immunoglobulin heavy chain transcriptional control element, animmunoglobulin κ light chain transcriptional control element, animmunoglobulin λ light chain transcriptional control element, or anycombination thereof.

In some embodiments, a nucleic acid sequence encoding an exogenous TdTis located at an immunoglobulin κ light chain locus, an immunoglobulin λlight chain locus, an immunoglobulin heavy chain locus, a RAG1 locus, ora RAG2 locus.

In some embodiments, the TdT is a human TdT. In some embodiments, theTdT is a short isoform of TdT (TdTS).

A human Igλ light chain sequence, in some embodiments, comprises geneticmaterial from (e.g., isolated or obtained from) or identical to a humanIgλ light chain locus, wherein the human Igλ light chain sequenceencodes an Ig light chain that comprises the encoded portion of thegenetic material from the human Igλ light chain locus. In someembodiments, a human Igλ light chain sequence as described hereincomprises at least one human Vλ gene segment and at least one human Jλgene segment, and one or more sequences necessary to promoterearrangement (e.g., recombination signal sequence(s)) of said at leastone human Vλ gene segment with said at least one human Jλ gene segmentto form a functional rearranged human Vλ-Jλ sequence that encodes ahuman Vλ domain. In many embodiments, a human Igλ light chain sequencecomprises a plurality of human Vλ and Jλ gene segments and one or moresequences necessary to promote rearrangement of said human Vλ genesegments with said human Jλ gene segments. In many embodiments, a humanIgλ light chain sequence comprises at least the coding sequences (e.g.,exons) of one or more human Vλ gene segments and at least the codingsequences (e.g., exons) of one or more human Jλ gene segments. In someembodiments, a human Igλ light chain sequence as described herein is agenomic sequence of a human Igλ light chain locus (e.g., isolated and/orcloned from a bacterial artificial chromosome) and contains a pluralityof human Vλ gene segments in germline configuration. In someembodiments, a human Igλ light chain sequence comprises human Vλ and Jλsequences (i.e., gene segments) in germline configuration (i.e., aplurality of human Vλ gene segments separated by intervening DNA thatincludes sequences necessary for and that promote recombination, and aplurality of Jλ gene segments separated by intervening DNA that incudessequences necessary for and that promote recombination).

In some embodiments, a human Igλ light chain sequence as describedherein is an engineered sequence and contains a plurality of human Jλgene segments in a configuration that is different than that whichappears in a human Igλ light chain locus in a human cell. In someembodiments, a human Igλ light chain sequence as described herein is anengineered sequence and contains a plurality of human Vλ and Jλ genesegments in a configuration that resembles or is similar to that whichappears in an Igκ light chain locus of a wild-type murine or human cell.In some embodiments, a human Igλ light chain sequence comprisesengineered human Jλ sequences (i.e., coding sequences of human Jλ genesegments made by de novo DNA synthesis that includes sequences necessaryfor and that promote recombination with one or more human Vλ genesegments). In some embodiments, a human Igλ light chain sequencecomprises Igκ and Igx sequences that naturally appear separately in Igκand Igλ genomic sequences, respectively. In some certain embodiments, ahuman Igλ light chain sequence comprises a Igκ sequence(s), inparticular, a Jκ region (i.e., a sequence that contains coding andnon-coding sequences that appear in a region containing a plurality ofJκ gene segments), that naturally appears in an Igκ light chain locusexcept that said Igκ sequence contains coding sequences of Jλ genesegments and Jλ 12RSS in the place of corresponding coding sequences ofJκ gene segments and Jκ 23RSS, respectively. In some certainembodiments, a human Igλ light chain sequence comprises a plurality ofJλ gene segments and Jλ 12RSS in the place of Jκ gene segments and Jκ23RSS of a Jκ region sequence. In various embodiments, intervening (orintergenic) DNA that includes sequences necessary for and that promoterecombination includes human Igκ and/or human Igλ genomic sequence(s).Alternatively, and in some embodiments, intervening (or intergenic) DNAthat includes sequences necessary for and that promote recombinationincludes murine Igκ and/or murine Igλ genomic sequence(s).

In some certain embodiments, a human Igλ light chain sequence is orcomprises a sequence that appears in the Drawing. In some embodiments, ahuman Igλ light chain sequence encodes, or is capable of encoding (e.g.,after rearrangement of human gene segments), a Vλ domain polypeptide,which Vλ domain polypeptide appears in an immunoglobulin, in particular,an immunoglobulin that is expressed by a human B cell. Non-humananimals, embryos, cells and targeting constructs for making non-humananimals, non-human embryos, and cells containing said human Igλ lightchain sequence in the place of a corresponding non-human Igκ light chainsequence (e.g., an endogenous rodent Igκ light chain locus) are alsoprovided.

In some embodiments, a human Igλ light chain sequence is inserted in theplace of a corresponding non-human Igκ light chain sequence within thegermline genome of a non-human animal. In some embodiments, a human Igλlight chain sequence is inserted upstream of a non-human Igλ light chainsequence (e.g., a non-human Igλ light chain constant region genesequence), which non-human Igλ light chain sequence is positioned in theplace of a non-human Igκ light chain sequence (e.g., a non-human Igκlight chain constant region gene sequence). In some embodiments, a humanIgκ light chain sequence is inserted in the midst of said human Igλlight chain sequence (i.e., between human Vλ and Jλ gene segments) sothat said human Igκ light chain sequence is juxtaposed by human Igλlight chain sequences.

In some embodiments, all or substantially all of the variable region ofa non-human Igκ light chain locus is replaced or substituted with one ormore human Igλ light chain sequences (as described herein), and said oneor more human Igλ light chain sequences are operably linked to anon-human or human Igλ light chain constant region gene. In someembodiments, a non-human Igκ light chain constant region gene is deletedor replaced in a non-human animal that includes a human Igλ light chainsequence as described herein. In one non-limiting example, in theinstance of an insertion of a human Igλ light chain sequence that isinserted into a non-human Igκ light chain locus, said insertion is madein manner to maintain the integrity of non-human Igκ light chainenhancer regions (or enhancer sequences) near the insertion point (e.g.,a non-human Igκ intronic enhancer and/or a non-human Igκ 3′ enhancer).Thus, such non-human animals have wild-type Igλ light chain enhancerregions (or enhancer sequences) operably linked to human and non-humanIgλ light chain sequences (e.g., human Vλ and Jλ gene segments, and anon-human Cλ region gene) or operably linked to human Igλ light chainsequences (e.g., human Vλ and Jλ gene segments, and a human Cλ regiongene). In some embodiments, a non-human Igκ light chain locus that isaltered, displaced, disrupted, deleted, replaced or engineered with oneor more human Igλ light chain sequences as described herein is a murineIgκ light chain locus. In some embodiments, one or more human Igλ lightchain sequences as described herein is inserted into one copy (i.e.,allele) of a non-human Igκ light chain locus of the two copies of saidnon-human Igκ light chain locus, giving rise to a non-human animal thatis heterozygous with respect to the human Igκ light chain sequence. Insome embodiments of a non-human animal that is heterozygous with respectto the human Igκ light chain sequence, the non-human animal includes oneor more human Igκ light chain sequences inserted into the other copy(i.e., allele) of the non-human Igκ light chain locus. In someembodiments, a non-human animal is provided that is homozygous for anIgκ light chain locus that includes one or more human Igλ light chainsequences as described herein.

In some embodiments, an engineered non-human Igκ light chain locus asdescribed herein comprises human Vλ and Jλ gene segments operably linkedto a non-human or human Igλ light chain constant region gene, whereinsaid non-human or human Igλ light chain constant region gene is locatedin the place of a non-human Igκ light chain constant region gene thatappears in a wild-type Igκ light chain locus of a non-human animal ofthe same species.

In some embodiments, one or more endogenous non-human Igλ light chainsequences (or portions thereof) of an endogenous non-human Igλ lightchain locus are not deleted. In some embodiments, one or more endogenousnon-human Igλ light chain sequences (or portions thereof) of anendogenous non-human Igλ light chain locus are deleted. In someembodiments, one or more endogenous non-human Igλ light chain sequences(e.g., V, J and/or C or any combination thereof) of an endogenousnon-human Igλ light chain locus is altered, displaced, disrupted,deleted or replaced so that said non-human Igλ light chain locus isfunctionally silenced. In some embodiments, one or more endogenousnon-human Igλ light chain sequences (e.g., V, J and/or C or anycombination thereof) of an endogenous non-human Igλ light chain locus isaltered, displaced, disrupted, deleted or replaced with a targetingvector so that said non-human Igλ light chain locus is functionallyinactivated (i.e., unable to produce a functional light chain of anantibody that is expressed and/or detectable in the antibody repertoireof the non-human animal). Guidance for inactivation of an endogenousnon-human Igλ light chain locus is provided in, e.g., U.S. Pat. No.9,006,511 (see, e.g., FIG. 2), which is incorporated herein by referencein its entirety.

In some embodiments, a non-human animal contains an engineered Igκ lightchain locus as described herein that is randomly integrated into itsgenome (e.g., as part of a randomly integrated human Igλ light chainsequence). Thus, such non-human animals can be described as having ahuman Igλ light chain transgene containing a plurality of human Vλ andJλ gene segments operably linked to a non-human or human Igλ light chainconstant region gene and non-human Igκ light chain enhancer regions (orenhancer sequences), so that that said human Vλ and Jλ gene segments arecapable of rearrangement and encoding an Ig light chain of an antibodyin the expressed repertoire of the non-human animal, which Ig lightchain includes a human Vλ domain and a non-human Cλ domain or which Iglight chain includes human Vλ and Cλ domains. An engineered Igκ lightchain locus or transgene as described herein can be detected using avariety of methods including, for example, PCR, Western blot, Southernblot, restriction fragment length polymorphism (RFLP), or a gain or lossof allele assay. In some embodiments, a non-human animal as describedherein is heterozygous with respect to an engineered Igκ light chainlocus as described herein. In some embodiments, a non-human animal asdescribed herein is hemizygous with respect to an engineered Igκ lightchain locus as described herein. In some embodiments, a non-human animalas described herein contains one or more copies of an engineered Igκlight chain locus or transgene as described herein. In some embodiments,a non-human animal as described herein contains an Igκ light chain locusas depicted in the Drawing.

The present disclosure recognizes that a non-human animal as describedherein will utilize human heavy chain, λ light chain, and κ light chainvariable region gene segments included in its genome in its antibodyselection and generation mechanisms (e.g., recombination and somatichypermutation). As such, in various embodiments, human immunoglobulinhuman heavy chain, λ light chain, and κ light chain variable domainsgenerated by non-human animals described herein are encoded by the humanheavy, λ light chain, and κ light chain variable region gene segmentsincluded in their genome or somatically hypermutated variants thereof,respectively.

In some embodiments, a non-human animal is provided whose genomecomprises an engineered immunoglobulin κ light chain locus, where thenon-human animal includes a B cell that includes a human heavy variableregion sequence, a human λ light chain variable region sequence, and/ora human κ light chain variable region sequence that is somaticallyhypermutated. In some embodiments, a human heavy variable regionsequence, a human λ light chain, and/or a human κ light chain variableregion sequence present in a B cell of a mouse of the present disclosurehas 1, 2, 3, 4, 5, or more somatic hypermutations. Those skilled in theart are aware of methods for identifying source gene segments in amature antibody sequence. For example, various tools are available toaid in this analysis, such as, for example, DNAPLOT, IMGT/V-QUEST,JOINSOLVER, SoDA, and Ab-origin.

The present disclosure provides, among other things, cells and tissuesfrom non-human animals described herein. In some embodiments, providedare splenocytes (and/or other lymphoid tissue) from a non-human animalas described herein. In some embodiments, provided is a B cell from anon-human animal as described herein. In some embodiments, provided is apro-B cell from a non-human animal as described herein. In someembodiments, provided is a pre-B cell from a non-human animal asdescribed herein. In some embodiments, provided is an immature B cellfrom a non-human animal as described herein. In some embodiments,provided is a mature naïve B cell from a non-human animal as describedherein. In some embodiments, provided is an activated B cell from anon-human animal as described herein. In some embodiments, provided is amemory B cell from a non-human animal as described herein. In someembodiments, provided is a B lineage lymphocyte from a non-human animalas described herein. In some embodiments, provided is plasma or a plasmacell from a non-human animal as described herein. In some embodiments,provided is a stem cell from a non-human animal as described herein. Insome embodiments, a stem cell is an embryonic stem cell. In someembodiments, provided is a germ cell from a non-human animal asdescribed herein. In some embodiments, a germ cell is an oocyte. In someembodiments, a germ cell is a sperm cell. In some embodiments, a spermcell from a non-human animal as described herein expresses one or moreADAM6 polypeptides, functional orthologs, functional homologs, orfunctional fragments thereof. In some embodiments, any cell or tissuefrom a non-human animal as described herein may be isolated. In someembodiments, provided is an isolated cell and/or an isolated tissue froma non-human animal as described herein. In some embodiments, a hybridomais provided, wherein the hybridoma is made with a B cell of a non-humananimal as described herein. In some embodiments, a hybridoma is madewith a B cell of a non-human animal that has been immunized with anantigen of interest. In some embodiments, a hybridoma is made with a Bcell of a non-human animal that expresses an antibody that binds (e.g.,specifically binds) to an epitope on an antigen of interest.

Any of the non-human animals as described herein may be immunized withone or more antigens of interest under conditions and for a timesufficient that the non-human animal develops an immune response to saidone or more antigens of interest. Those skilled in the art are aware ofmethods for immunizing non-human animals. An exemplary, non-limitingmethod for immunizing non-human animals can be found in US2007/0280945A1, incorporated herein by reference in its entirety.

The present disclosure provides, among other things, immunized non-humananimals as described herein, and cells and tissues isolated from thesame. In some embodiments, a non-human animal described herein producesa population of B cells in response to immunization with an antigen thatincludes one or more epitopes. In some embodiments, a non-human animalproduces a population of B cells that express antibodies that bind(e.g., specifically bind) to one or more epitopes of antigen ofinterest. In some embodiments, antibodies expressed by a population of Bcells produced in response to an antigen include a heavy chain having ahuman heavy chain variable domain encoded by a human heavy chainvariable region sequence and/or a lambda light chain having a humanlambda light chain variable domain encoded by a human lambda light chainvariable region sequence as described herein. In some embodiments,antibodies expressed by a population of B cells produced in response toan antigen include (i) a heavy chain having a human heavy chain variabledomain encoded by a human heavy chain variable region sequence, (ii) alambda light chain having a human lambda light chain variable domainencoded by a human lambda light chain variable region sequence asdescribed herein, (iii) a kappa light chain having a human kappa lightchain variable domain encoded by a human kappa light chain variableregion sequence as described herein, or (iv) any combination thereof.

In some embodiments, a non-human animal produces a population of B cellsthat express antibodies that bind to one or more epitopes of antigen ofinterest, where antibodies expressed by the population of B cellsproduced in response to an antigen include: (i) a heavy chain having ahuman heavy chain variable domain encoded by a human heavy chainvariable region sequence, (ii) a lambda light chain having a humanlambda light chain variable domain encoded by a human lambda light chainvariable region sequence as described herein, (iii) a kappa light chainhaving a human kappa light chain variable domain encoded by a humankappa light chain variable region sequence as described herein, or (iv)any combination thereof. In some embodiments, a human heavy chainvariable region sequence, a human λ light chain variable regionsequence, and/or a human κ light chain variable region sequence asdescribed herein is somatically hypermutated. In some embodiments, atleast about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90% of the B cells in a population of B cellsproduced in response to an antigen include a human heavy chain variableregion sequence, a human λ light chain variable region sequence, and/ora human κ light chain variable region sequence that is somaticallyhypermutated.

In some embodiments, non-human animals provided herein, in theirgermline genome, (1) include an engineered endogenous immunoglobulin κlight chain locus comprising (a) one or more human Vλ gene segments, (b)one or more human Jλ gene segments, and (c) a Cλ gene, where the one ormore human Vλ gene segments and one or more human Jλ gene segments areoperably linked to the Cλ gene, (2) lack a rodent Cκ gene at theengineered endogenous immunoglobulin κ locus, and (3) include anengineered endogenous immunoglobulin κ light chain locus comprising (a)one or more human Vκ gene segments, (b) one or more human Jκ genesegments, and (c) a Cκ gene, where the one or more human Vκ genesegments and one or more human Jκ gene segments are operably linked tothe Cκ gene. In some embodiments, the percentage of light chains insplenocytes (e.g., as detected or observed, e.g., by flow cytometry(see, e.g., Example 3)) of such non-human animals that are λ lightchains is at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, or at least 75%. Insome embodiments, the percentage of light chains in splenocytes (e.g.,as detected or observed, e.g., by flow cytometry (see, e.g., Example 3))of such non-human animals that are λ light chains is between 35-80%,between 35-75%, between 40-80%, between 40-75%, between 50-80%, between50-75%, between 55-80%, between 55-75%, between 60-80%, or between60-75%. In some embodiments, the percentage of light chains insplenocytes (e.g., as detected or observed, e.g., by flow cytometry(see, e.g., Example 3)) of such non-human animals that are κ lightchains is at most 65%, at most 60%, at most 55%, at most 50%, at most45%, at most 40%, or at most 35%. In some embodiments, the percentage oflight chains in splenocytes (e.g., as detected or observed, e.g., byflow cytometry (see, e.g., Example 3)) of such non-human animals thatare κ light chains is between 20-65%, between 25-65%, between 20-60%,between 25-60%, between 20-55%, between 25-55%, between 20-50%, between25-50%, between 20-45%, between 25-45%, between 20-40%, or between25-40%. In some embodiments, the ratio of κ:λ light chains insplenocytes (e.g., as detected or observed, e.g., by flow cytometry(see, e.g., Example 3)) of such non-human animals is between 0.5:1 and3:1, 0.65:1 and 3:1, between 0.8:1 and 3:1, between 1:1 and 3:1, between1.2:1 and 3:1, between 1:1 and 2.3:1, between 1.1:1 and 1.8:1, between1.2:1 and 2.3:1, or between 1.2:1 and 1.8:1.

Methods of Making Provided Non-Human Animals

Compositions and methods for making non-human animals whose germlinegenome comprises an engineered Igκ light chain locus that includes oneor more human Igλ light chain sequences (e.g., human Vλ and Jλ genesegments) in the place of non-human Igκ light chain sequences, includinghuman Igλ light chain encoding sequences that include specificpolymorphic forms of human Vλ and Jλ segments (e.g., specific V and/or Jalleles or variants) are provided, including compositions and methodsfor making non-human animals that express antibodies comprising Igλlight chains that contain human variable regions and non-human or humanconstant regions, assembled from an Igκ light chain locus that containshuman Vλ and Jλ gene segments operably linked to a non-human or humanIgλ light chain constant region gene, which non-human or human Igλ lightchain constant region gene is located in the place of a non-human Igκlight chain constant region gene that normally appears in a wild-typenon-human Igκ light chain locus. In some embodiments, compositions andmethods for making non-human animals that express such antibodies underthe control of an endogenous Igκ enhancer(s) and/or an endogenous Igκregulatory sequence(s) are also provided. In some embodiments,compositions and methods for making non-human animals that express suchantibodies under the control of a heterologous Igκ enhancer(s) and/or aheterologous Igκ regulatory sequence(s) are also provided.

Methods described herein include inserting human Vλ and Jλ sequencesencoding human Vλ domains upstream of a non-human or human Igλ lightchain constant region gene (e.g., a murine or human Cλ region gene),which non-human or human Igλ light chain constant region gene is locatedin the place of a non-human Igκ light chain constant region gene thatnormally appears in a wild-type non-human Igκ light chain locus, so thatan antibody is expressed, which antibody is characterized by thepresence of a light chain that contains a human Vλ domain and anon-human Cλ domain (e.g., a rodent Cλ domain) or by the presence of alight chain that contains human Vλ and non-human Cλ domains (e.g., oneor more rodent Cλ domains), and is expressed both on the surface of Bcells and in the blood serum of a non-human animal.

In some embodiments, methods include insertion of genetic material thatcontains human Vλ and Jλ gene segments into an Igκ light chain locus(e.g., a wild-type, modified or engineered Igκ light chain locus). Insome certain embodiments, methods include insertion of genetic materialthat contains human Jλ gene segments into an Igκ light chain locus of amodified or engineered strain. In some embodiments, genetic materialthat contains human Igλ light chain sequences can be engineered orgenomic (e.g., cloned from a bacterial artificial chromosome). In someembodiments, genetic material that contains human Igλ light chainsequences can be designed from published sources and/or bacterialartificial chromosomes so that said genetic material contains human Vλand Jλ segments in an orientation that is different from that whichappears in a human Igλ light chain locus yet said genetic material stillcontains sequences to support rearrangement of said human Vλ and Jλsegments to encode a functional human Vλ domain of an Ig light chain. Togive but one example, genetic material corresponding to a plurality ofhuman Vλ and Jλ gene segments can be designed using the guidanceprovided herein to construct a human Igλ light chain sequence thatcontains human Vλ and Jλ segments in an order and/or arrangement that isdifferent than that which appears in a human Igλ light chain locus of ahuman cell (e.g., an arrangement that resembles or is similar to a humanor rodent Igκ light chain locus, such as, a series of V gene segments,followed 3′ by intervening DNA, followed 3′ by a series of J genesegments). In such an example, genetic content of human Vλ and Jλ genesegments would be equivalent to the corresponding segments in a humancell, however, the order and arrangement would be different. Whenconstructing an engineered Igκ light chain locus for generation of anon-human animal as described herein, the requisite recombination signalsequences can be configured so that the human V and J gene segments cancorrectly rearrange and form a functional human Vλ domain. Guidance forgermline configuration of human Vλ and Jλ gene segments and sequencesnecessary for proper recombination can be found in, e.g., MolecularBiology of B Cells, London: Elsevier Academic Press, 2004, Ed. Honjo,T., Alt, F. W., Neuberger, M. Chapters 4 (pp. 37-59) and 5 (61-82);incorporated herein by reference in their entireties.

In some embodiments, methods include multiple insertions in a single EScell clone. In some embodiments, methods include sequential insertionsmade in a successive ES cell clones. In some embodiments, methodsinclude a single insertion made in an engineered ES cell clone.

In some embodiments, methods include DNA insertion(s) upstream of amurine Cλ1 gene (or human Cλ2 gene) so that said DNA insertion(s) isoperably linked to said murine Cλ1 gene (or human Cλ2 gene), which DNAinsertion(s) comprise human Vλ gene segments Vλ4-69, Vλ8-61, Vλ4-60,Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23,Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9,Vλ2-8, Vλ4-3, Vλ3-1 or any combination thereof, and human Jλ genesegments Jλ1, Jλ2, Jλ3, Jλ6, Jλ7, or any combination thereof, and whichmurine Cλ1 gene (or human Cλ2 gene) is located in the place of a murineCκ gene of an endogenous Igκ light chain locus.

In some embodiments, methods include DNA insertion(s) downstream of ahuman Vλ3-1 gene segment and upstream of a non-human Igκ intronicenhancer region (or enhancer sequence) of an engineered Igκ light chainlocus, so that said DNA insertion(s) is operably linked to a murine Cλ1gene (or human Cλ2 gene), which DNA insertion(s) comprises a human Igκgenomic sequence that naturally appears between a human Vκ4-1 genesegment and a human Jκ1 gene segment of a human Igκ light chain locus,and one or more human Jλ gene segments (e.g., one, two, three, four,five, six or seven), which murine Cλ1 gene (or human Cλ2 gene) islocated in the place of a murine Cκ gene of an endogenous non-human Igκlight chain locus. In some certain embodiments, methods include DNAinsertion(s) between a human Vλ3-1 gene segment and a non-human Igκintronic enhancer, which DNA insertion(s) includes human Vκ-Jκ sequencethat naturally appears between human Vκ4-1 and Jκ1 gene segments of ahuman Igκ light chain locus and five human Jλ gene segments (e.g., Jλ1,Jλ2, Jλ3, Jλ6 and Jλ7). In various embodiments, DNA insertion(s)including human Jλ gene segments comprises human Jκ genomic DNA withcoding sequences of human Jλ gene segments and human Jλ 12RSS.

Insertion of additional human Vλ and Jλ segments may be achieved usingmethods described herein to further supplement the diversity of anengineered Igλ light chain locus. For example, in some embodiments,methods can include insertion of about 270 kb of DNA upstream of amurine Cλ1 gene (or human Cλ2 gene) of an engineered Igκ light chainlocus so that said DNA is operably linked to said murine Cλ1 gene (orhuman Cλ2 gene), which DNA includes human Vλ gene segments Vλ10-54,Vλ6-57, Vλ4-60, Vλ8-61 and Vλ4-69. In such embodiments, said DNA isinserted upstream of a human Vλ5-52 gene segment that is operably linkedto a murine Cλ1 gene (or human Cλ2 gene) of an engineered Igκ lightchain locus, which DNA includes human Vλ gene segments Vλ10-54, Vλ6-57,Vλ4-60, Vλ8-61 and Vλ4-69. In some certain embodiments, said DNAincludes a human VpreB gene. Additional human Vλ gene segments describedabove may be cloned directly from commercially available BAC clones andarranged in smaller DNA fragment using recombinant techniques describedherein or otherwise known in the art. Alternatively, additional human Vλgene segments described above can be synthesized as an engineered DNAfragment and added to an engineered Igκ light chain locus as describedabove using molecular biology techniques known in the art. Likewise,additional human Jλ gene segments may be obtained from commerciallyavailable BAC clones or synthesized directly from published sequences.An exemplary illustration that shows an engineered Igκ light chain locusof non-human animals as described herein is set forth in FIG. 2B or 4B.

Where appropriate, a human Igλ light chain sequence (i.e., a sequencecontaining human Vλ and Jλ gene segments) encoding a human Vλ domain mayseparately be modified to include codons that are optimized forexpression in a non-human animal (e.g., see U.S. Pat. Nos. 5,670,356 and5,874,304). Codon optimized sequences are engineered sequences, andpreferably encode the identical polypeptide (or a biologically activefragment of a full-length polypeptide which has substantially the sameactivity as the full-length polypeptide) encoded by the non-codonoptimized parent polynucleotide. In some embodiments, a human Igλ lightchain sequence encoding a human Vλ domain may separately include analtered sequence to optimize codon usage for a particular cell type(e.g., a rodent cell). For example, the codons of each nucleotidesequence to be inserted into the genome of a non-human animal (e.g., arodent) may be optimized for expression in a cell of the non-humananimal. Such a sequence may be described as a codon-optimized sequence.

Insertion of nucleotide sequences encoding human Vλ domains employs aminimal modification of the germline genome of a non-human animal asdescribed herein and results in expression of antibodies comprisinglight chains having human Vλ domains, which human Vλ domains areexpressed from endogenous engineered Igκ light chain loci. Methods forgenerating engineered non-human animals, including knockouts andknock-ins, are known in the art (see, e.g., Gene Targeting: A PracticalApproach, Joyner, ed., Oxford University Press, Inc., 2000; incorporatedherein by reference in its entirety). For example, generation ofgenetically engineered rodents may optionally involve disruption of thegenetic loci of one or more endogenous rodent genes (or gene segments)and introduction of one or more heterologous genes (or gene segments ornucleotide sequences) into the rodent genome, in some embodiments, atthe same location as an endogenous rodent gene (or gene segments). Insome embodiments, nucleotide sequences encoding human Vλ domains areintroduced upstream of a murine or human Igλ light chain constant regiongene of a randomly inserted engineered light chain transgene in thegermline genome of a rodent. In some embodiments, nucleotide sequencesencoding human Vλ domains are introduced upstream of a murine or humanIgλ light chain constant region gene of an endogenous Igκ light chainlocus in the germline genome of a rodent; in some certain embodiments,an endogenous Igκ light chain locus is altered, modified, or engineeredto contain human Igλ gene segments (e.g., human V and J) operably linkedto a mouse Cλ1 gene or operably linked to a human Cλ2 gene.

Schematic illustrations (not to scale) of exemplary methods forconstructing an engineered Igκ light chain locus as described herein areprovided in FIGS. 1A, 1B, 2A, 2B, 3, 4A and 4B. In particular, FIGS. 1Aand 1B sets forth an exemplary strategy for construction of anengineered Igκ light chain locus characterized by insertion ofnucleotide sequences containing a plurality of human Vλ and Jλ genesegments. As illustrated in FIGS. 1A and 1B, a DNA fragment containing ahuman Vκ-Jκ intergenic region (see U.S. Pat. Nos. 9,006,511, 9,012,717,9,029,628, 9,035,128, 9,066,502, 9,150,662 and 9,163,092) and engineeredfragment containing a set of human Jλ gene segments (e.g., human Jλ1,Jλ2, Jλ3, Jλ6 and Jλ7) is operably linked to a rodent Igκ intronicenhancer region (or enhancer sequence) via a series of steps usingvarious molecular biology techniques described in Example 1. Thisengineered fragment is also engineered to contain a rodent Igλ lightchain constant region that is operably linked to the human Jλ genesegments. Selection cassettes (e.g., Neomycin and Hygromycin) areincluded in the targeting vector to allow for selection of positiveclones in bacteria and mammalian cells (e.g., embryonic stem cells). Asillustrated a Neomycin resistance gene is flanked by lox2372site-specific recombination sites (lox) and positioned between the humanVκ-Jκ region and the set of human Jλ gene segments, while the Hygromycinselection cassette is flanked by loxP site-specific recombination sitesand positioned 3′ of the rodent Igλ light chain constant region (mCλ1)gene. The DNA fragment is then combined with a DNA fragment containing arodent Igκ light chain 3′ enhancer to create the final targeting vector(FIG. 1B). The resulting targeting vector (construct G) is linearizedand electroporated into rodent embryonic stem (ES) cells to create arodent whose germline genome comprises the engineered Igκ light chainlocus. As described in the examples section below, the rodent ES cellsemployed in electroporation of the targeting vector contained anengineered Igκ light chain locus as previously described in U.S. Pat.Nos. 9,006,511, 9,012,717, 9,029,628, 9,035,128, 9,066,502, 9,150,662and 9,163,092; incorporated herein by reference in their entireties.Homologous recombination with the targeting vector as depicted in FIG. 3results in an engineered Igκ light chain locus characterized by aplurality of human Vλ and Jλ gene segments operably linked to a murineCλ1 gene, which murine Cλ1 gene is located in place of a murine Cκ genethat naturally appears in a wild-type Igκ light chain locus. The humanJλ gene segments are uniquely engineered into a sequence that naturallyappears in a genomic human Jκ region yet has human Jλ coding sequencesand associated 12RSS in the place of human Jκ coding sequences andassociated 23RSS. Positive rodent ES cell clones are confirmed usingscreening methods described herein and/or known in the art. Anyremaining selection cassette may be deleted as desired viarecombinase-mediated deletion (see Example 2).

Alternatively, a human Cλ gene may be employed in a targeting vectorinstead of a mouse Cλ gene. To give but one example, FIG. 3 illustratesa targeting vector that was constructed in a similar manner as describedabove except that a sequence encoding a human Cλ2 gene was engineeredinto the targeting vector and in operable linkage with five human Jλgene segments. Using such an approach provides an added benefit indeveloping human antibody therapeutics as DNA encoding the variable andconstant regions of light chains may be isolated together, therebyeliminating any subsequent cloning step linking to a human light chainconstant region for the preparation of fully-human antibodies.

Targeting vectors for constructing an engineered Igκ light chain locusas described herein may be incorporated into the germline genome of anon-human cell (e.g., a rodent embryonic stem cell). In someembodiments, targeting vectors as described herein are incorporated intoa wild-type Igλ light chain locus in the germline genome of a non-humancell that further contains human V_(H), D_(H) and J_(H) genomic DNA(e.g., containing a plurality of human V_(H), D_(H) and J_(H) genesegments) operably linked with one or more immunoglobulin heavy chainconstant region genes (e.g., see U.S. Pat. Nos. 8,502,018, 8,642,835,8,697,940 and 8,791,323, each of which is incorporated herein byreference in its entirety). In some embodiments, targeting vectors asdescribed herein are incorporated into a modified or engineeredimmunoglobulin κ light chain locus in the germline genome of a non-humancell that further contains human V_(H), D_(H) and J_(H) genomic DNA(e.g., containing a plurality of human V_(H), D_(H) and J_(H) genesegments) operably linked with one or more immunoglobulin heavy chainconstant region genes (e.g., see U.S. Pat. Nos. 8,502,018, 8,642,835,8,697,940, 8,791,323, 9,006,511, 9,012,717, 9,029,628, 9,035,128,9,066,502, 9,150,662 and 9,163,092, each of which is incorporated hereinby reference in its entirety).

A targeting vector is introduced into rodent (e.g., mouse) embryonicstem cells by electroporation so that the sequence contained in thetargeting vector results in the capacity of a non-human cell ornon-human animal (e.g., a mouse) that expresses antibodies having lightchains that include human Vλ domains and non-human or human Cλ domains,and which light chains are expressed from an endogenous engineeredimmunoglobulin κ light chain locus. As described herein, a geneticallyengineered rodent is generated where an engineered immunoglobulin κlight chain locus has been created in the germline genome of the rodent(e.g., an endogenous immunoglobulin κ light chain locus containing ahuman Igλ light chain sequence (i.e., a plurality of human Vλ and Jλgene segments) operably linked to a rodent or human Cλ gene in the placeof an endogenous rodent Cκ gene). Antibodies are expressed on thesurface of rodent B cells and in the serum of said rodent, whichantibodies are characterized by light chains having human Vλ domains andnon-human or human Cλ domains. When an endogenous immunoglobulin κ lightchain locus in the germline genome of the rodent is not targeted by thetargeting vector, an engineered immunoglobulin κ light chain transgeneis preferably inserted at a location other than that of an endogenousrodent immunoglobulin κ light chain locus (e.g., randomly insertedtransgene).

Creation of an engineered immunoglobulin κ light chain locus in anon-human animal as described above provides an engineered rodent strainthat produces antibodies that include immunoglobulin λ light chainsexpressed from such an engineered immunoglobulin κ light chain locushaving a human Vλ domain and a non-human or human Cλ domain. Leveragedwith the presence of an engineered immunoglobulin heavy chain locus thatincludes a plurality of human V_(H), D_(H) and J_(H) gene segmentsoperably linked to immunoglobulin heavy chain constant region genes, anengineered rodent strain that produces antibodies and antibodycomponents for the development of human antibody-based therapeutics iscreated. Thus, a single engineered rodent strain is realized that hasthe capacity to provide an alternative in vivo system for exploitinghuman Vλ domains for the development of new antibody-based medicines totreat human disease.

In some embodiments, a method of making a non-human animal whosegermline genome comprises an engineered endogenous immunoglobulin κlight chain locus is provided, the method comprising (a) introducing aDNA fragment into a non-human embryonic stem cell, said DNA fragmentcomprising a nucleotide sequence that includes (i) one or more human Vλgene segments, (ii) one or more human Jλ gene segments and (iii) a Cλgene (e.g., non-human or human), wherein (i)-(iii) are operably linked,and wherein the nucleotide sequence further comprises an immunoglobulink light chain sequence between (i) and (ii), (b) obtaining the non-humanembryonic stem cell generated in (a); and (c) creating a rodent usingthe rodent embryonic stem cell of (b).

In some embodiments, a method of making a non-human animal whosegermline genome comprises an engineered endogenous immunoglobulin κlight chain locus is provided, the method comprising (a) introducing aDNA fragment into a non-human embryonic stem cell, said DNA fragmentcomprising a nucleotide sequence that includes one or more human Jλ genesegments, one or more non-human immunoglobulin κ light chain enhancers,and a non-human or human Cλ gene, which human Jλ gene segments areoperably linked to said one or more non-human immunoglobulin κ lightchain enhancers and said non-human or human Cλ gene, (b) obtaining thenon-human embryonic stem cell generated in (a); and (c) creating arodent using the rodent embryonic stem cell of (b).

In some embodiments, a method of making a non-human animal whosegermline genome comprises an engineered endogenous immunoglobulin κlight chain locus, which engineered endogenous immunoglobulin κ lightchain locus comprises insertion of one or more human Vλ gene segments,one or more human Jλ gene segments and a non-human or human Cλ gene,which human Vλ and Jλ gene segments are operably linked to saidnon-human or human Cλ gene, and which non-human or human Cλ gene isinserted in the place of a non-human Cκ gene at the endogenousimmunoglobulin κ locus, is provided, the method comprising modifying thegermline genome of a non-human animal so that it comprises an engineeredendogenous immunoglobulin κ light chain locus that includes insertion ofone or more human Vλ gene segments, one or more human Jλ gene segmentsand a non-human or human Cλ gene, which human Vλ and Jλ gene segmentsare operably linked to said non-human or human Cλ gene, and whichnon-human or human Cλ gene is inserted in the place of a non-human Cκgene at the endogenous immunoglobulin κ locus.

In some embodiments of a method of making a non-human animal, one ormore human Vλ gene segments includes at least 24, at least 34, at least52, at least 61, or at least 70 human Vλ gene segments. In someembodiments of a method of making a non-human animal, one or more humanVλ gene segments include 39 human Vλ gene segments. In some certainembodiments of a method of making a non-human animal, one or more humanVλ gene segments include human Vλ4-69, Vλ8-61, Vλ4-60, Vλ6-57, Vλ10-54,Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40,Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19,Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, Vλ3-1 orany combination thereof. In some certain embodiments, one or more humanVλ gene segments include human non-coding DNA that naturally appearsadjacent to the relevant human Vλ gene segments in an endogenous human λlight chain locus.

In some embodiments of a method of making a non-human animal, one ormore human Jλ gene segments includes at least 1, at least 2, at least 3,at least 4 or at least 5 human Jλ gene segments. In some embodiments ofa method of making a non-human animal, one or more human Jλ genesegments includes 5 human Jλ gene segments. In some embodiments of amethod of making a non-human animal, one or more human Jλ gene segmentscomprise human Jλ1, Jλ2, Jλ3, Jλ6, Jλ7, or any combination thereof. Insome certain embodiments, one or more human Jλ gene segments includehuman non-coding DNA, in whole or in part, that naturally appearsadjacent to the relevant human Jλ gene segments in an endogenous human λlight chain locus. In some embodiments, one or more human Jλ genesegments include human non-coding DNA that naturally appears adjacent toa human Jκ1-Jκ5 in an endogenous human κ light chain locus.

In some embodiments of a method of making a non-human animal, a DNAfragment includes intergenic DNA that contains non-coding immunoglobulinDNA (e.g., DNA that naturally appears between the coding sequence of twoV gene segments, a V and J gene segment or between two J gene segments).In many embodiments, said non-coding immunoglobulin DNA is non-codingimmunoglobulin light chain DNA (e.g., human or murine). In someembodiments, non-coding immunoglobulin light chain DNA is immunoglobulinκ light chain DNA, immunoglobulin λ light chain DNA or combinationsthereof.

In some embodiments of a method of making a non-human animal, a DNAfragment further comprises one or more selection markers. In someembodiments of a method of making a non-human animal, a DNA fragmentfurther comprises one or more site-specific recombination sites. In somecertain embodiments of a method of making a non-human animal, a DNAfragment further comprises one or more sets of site-specificrecombination sites that recombine with the same recombinase. In somecertain embodiments of a method of making a non-human animal, a DNAfragment further comprises one or more sets of site-specificrecombination sites that recombine with different recombinases.

In some embodiments of a method of making a non-human animal, a DNAfragment comprises an engineered sequence that includes immunoglobulin κlight chain sequence and immunoglobulin λ light chain sequence togetherin a continuous sequence. In some embodiments of a method of making anon-human animal, a DNA fragment comprises an engineered sequence thatincludes immunoglobulin κ light chain sequence and immunoglobulin λlight chain sequence together in a single sequence yet interrupted by anon-immunoglobulin sequence (e.g., a recombination signal sequence, aresistance gene, and combinations thereof). In some certain embodimentsof a method of making a non-human animal, an engineered sequenceincludes portions of a Jκ region and portions of a Jλ region. In someembodiments, an engineered sequence includes portions of a human Jκregion and portions of a human Jλ region. In some certain embodiments,portions of a human Jκ region include non-coding sequences of a human Jκregion that naturally appear in a human immunoglobulin κ light chainlocus of a human cell. In some certain embodiments, portions of a humanJλ region include coding sequences and recombination signal sequences(RSS) of one or more human Jλ gene segments. In some certain embodimentsof a method of making a non-human animal, a DNA fragment comprises anengineered sequence that is characterized, in some embodiments, by thepresence of coding sequences and recombination signal sequences (RSS) ofone or more human Jλ gene segments that positionally replace orsubstitute (i.e., positioned in the place of) the corresponding codingsequences and recombination signal sequences (RSS) of human Jκ genesegments so that said coding sequences and recombination signalsequences (RSS) of said one or more human Jλ gene segments are within,adjacent to, contiguous with or juxtaposed by said non-coding sequencesof said one or more human Jκ gene segments.

In some embodiments of a method of making a non-human animal, a DNAfragment is introduced into a non-human embryonic stem cell whosegermline genome comprises one or more engineered immunoglobulin loci(e.g., immunoglobulin heavy chain, immunoglobulin κ light chain,immunoglobulin λ light chain, and combinations thereof). In some certainembodiments, engineered immunoglobulin loci are endogenous engineeredimmunoglobulin loci.

In some embodiments of a method of making a non-human animal, a DNAfragment is introduced into a non-human embryonic stem cell whosegermline genome comprises an endogenous immunoglobulin heavy chain locuscomprising insertion of one or more human V_(H) gene segments, one ormore human D_(H) gene segments and one or more human J_(H) genesegments, which human V_(H), D_(H) and J_(H) gene segments are operablylinked to a non-human immunoglobulin heavy chain constant region.

In some embodiments of a method of making a non-human animal, a DNAfragment is introduced into a non-human embryonic stem cell whosegermline genome comprises an endogenous immunoglobulin κ light chainlocus comprising insertion of one or more human Vλ and one or more humanJλ gene segments, which human Vλ and Jλ gene segments are operablylinked to a non-human immunoglobulin κ light chain constant region gene.In some certain embodiments of a method of making a non-human animal, aDNA fragment is introduced into a non-human embryonic stem cell whosegermline genome comprises an endogenous immunoglobulin κ light chainlocus comprising insertion of one or more human Vλ and one or more humanJλ gene segments, and a human immunoglobulin κ light chain sequencepositioned, placed or located between said one or more human Vλ genesegments and said one or more human Jλ gene segments, which human Vλ andJλ gene segments are operably linked to a non-human immunoglobulin κlight chain constant region gene.

In some embodiments of a method of making a non-human animal, modifyingthe germline genome of a non-human animal so that it comprises anengineered immunoglobulin κ light chain locus is carried out in anon-human embryonic stem cell whose germline genome comprises anendogenous immunoglobulin heavy chain locus comprising insertion of oneor more human V_(H) gene segments, one or more human D_(H) gene segmentsand one or more human J_(H) gene segments, which human V_(H), D_(H) andJ_(H) gene segments are operably linked to a non-human immunoglobulinheavy chain constant region.

In some embodiments of a method of making a non-human animal, modifyingthe germline genome of a non-human animal so that it comprises anengineered immunoglobulin κ light chain locus is carried out in anon-human embryonic stem cell whose germline genome comprises anendogenous immunoglobulin κ light chain locus comprising insertion ofone or more human Vλ and one or more human Jλ gene segments, which humanVλ and Jλ gene segments are operably linked to a non-humanimmunoglobulin κ light chain constant region gene. In some embodimentsof a method of making a non-human animal, modifying the germline genomeof a non-human animal so that it comprises an engineered immunoglobulinκ light chain locus is carried out in a non-human embryonic stem cellwhose germline genome comprises an endogenous immunoglobulin κ lightchain locus comprising insertion of one or more human Vλ and one or morehuman Jλ gene segments, and a human immunoglobulin κ light chainsequence positioned, placed or located between said one or more human Vλgene segments and said one or more human Jλ gene segments, which humanVλ and Jλ gene segments are operably linked to a non-humanimmunoglobulin κ light chain constant region gene.

In some embodiments of a method of making a non-human animal, insertionof one or more human V_(H) gene segments, one or more human D_(H) genesegments and one or more human J_(H) gene segments includes humannon-coding DNA that naturally appears adjacent to the human V_(H) genesegments, human non-coding DNA that naturally appears adjacent to thehuman D_(H) gene segments and human non-coding DNA that naturallyappears adjacent to the human J_(H) gene segments in an endogenous humanimmunoglobulin locus.

In some embodiments, a non-human animal made, generated, produced,obtained or obtainable from a method as described herein is provided.

In some embodiments, the genome of a non-human animal as describedherein further comprises one or more human immunoglobulin heavy variableregions as described in U.S. Pat. Nos. 8,502,018, 8,642,835, 8,697,940and 8,791,323, each of which is incorporated herein by reference in itsentirety. Alternatively, an engineered immunoglobulin κ light chainlocus as described herein can be engineered into an embryonic stem cellof a different modified strain such as, e.g., a VELOCIMMUNE® strain(see, e.g., U.S. Pat. Nos. 8,502,018 and/or 8,642,835; incorporatedherein by reference in their entireties). Homozygosity of the engineeredIgκ light chain locus as described herein can subsequently be achievedby breeding. Alternatively, in the case of a randomly insertedengineered immunoglobulin κ light chain transgene (described above),rodent strains can be selected based on, among other things, expressionof human Vλ domains from the transgene. In some embodiments, aVELOCIMMUNE® mouse can be a VELOCIMMUNE® 1 (VI-1) mouse, which includeseighteen human V_(H) gene segments, all of the human D_(H) genesegments, and all of the J_(H) gene segments. A VI-1 mouse can alsoinclude sixteen human Vκ gene segments and all of the human Jκ genesegments. In some embodiments, a VELOCIMMUNE® mouse can be aVELOCIMMUNE® 2 (VI-2) mouse, which includes thirty-nine human V_(H) genesegments, all of the human D_(H) gene segments, and all of the J_(H)gene segments. A VI-2 mouse can also include human thirty Vκ genesegments and all of the human Jκ gene segments. In some embodiments, aVELOCIMMUNE® mouse can be a VELOCIMMUNE® 3 (VI-3) mouse, which includeseighty human V_(H) gene segments, all of the human D_(H) gene segments,and all of the J_(H) gene segments. A VI-3 mouse can also include humanforty Vκ gene segments and all of the human Jκ gene segments.

Alternatively, and/or additionally, in some embodiments, the germlinegenome of a non-human animal as described herein further comprises adeleted, inactivated, functionally silenced or otherwise non-functionalendogenous immunoglobulin λ light chain locus. Genetic modifications todelete or render non-functional a gene or genetic locus may be achievedusing methods described herein and/or methods known in the art.

A genetically engineered founder non-human animal can be identifiedbased upon the presence of an engineered Igκ light chain locus in itsgermline genome and/or expression of antibodies having a human Vλ domainand a non-human or human Cλ domain in tissues or cells of the non-humananimal. A genetically engineered founder non-human animal can then beused to breed additional non-human animals carrying the engineeredimmunoglobulin κ light chain locus thereby creating a cohort ofnon-human animals each carrying one or more copies of an engineeredimmunoglobulin κ light chain locus. Moreover, genetically engineerednon-human animals carrying an engineered immunoglobulin κ light chainlocus as described herein can further be bred to other geneticallyengineered non-human animals carrying other transgenes (e.g., humanimmunoglobulin genes) or engineered immunoglobulin loci as desired.

Genetically engineered non-human animals may also be produced to containselected systems that allow for regulated, directed, inducible and/orcell-type specific expression of the transgene or integratedsequence(s). For example, non-human animals as described herein may beengineered to contain one or more sequences encoding a human Vλ domainof an antibody that is/are conditionally expressed (e.g., reviewed inRajewski, K. et al., 1996, J. Clin. Invest. 98(3):600-3, incorporatedherein by reference in its entirety). Exemplary systems include theCre/loxP recombinase system of bacteriophage P1 (see, e.g., Lakso, M. etal., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:6232-6, incorporated hereinby reference in its entirety) and the FLP/Frt recombinase system of S.cerevisiae (O'Gorman, S. et al, 1991, Science 251:1351-5, incorporatedherein by reference in its entirety). Such animals can be providedthrough the construction of “double” genetically engineered animals,e.g., by mating two genetically engineered animals, one containing atransgene comprising a selected modification (e.g., an engineered Igκlight chain locus as described herein) and the other containing atransgene encoding a recombinase (e.g., a Cre recombinase).

Non-human animals as described herein may be prepared as describedabove, or using methods known in the art, to comprise additional human,humanized or otherwise engineered genes, oftentimes depending on theintended use of the non-human animal. Genetic material of such human,humanized or otherwise engineered genes may be introduced through thefurther alteration of the genome of cells (e.g., embryonic stem cells)having the genetic modifications or alterations as described above orthrough breeding techniques known in the art with other geneticallymodified or engineered strains as desired. In some embodiments,non-human animals as described herein are prepared to further comprisehuman IgH and/or Igκ light chain genes or gene segments (see e.g.,Murphy, A. J. et al., (2014) Proc. Natl. Acad. Sci. U.S.A.111(14):5153-5158; U.S. Pat. Nos. 8,502,018, 8,642,835, 8,697,940 and8,791,323; 8,791,323; and U.S. Patent Application Publication No.2013/0096287 A1; each of which is incorporated herein by reference inits entirety).

In some embodiments, non-human animals as described herein may beprepared by introducing a targeting vector described herein into a cellfrom a modified or engineered strain. For example, a targeting vector asdescribed herein may be introduced into a VELOCIMMUNE® mouse.VELOCIMMUNE® mice express antibodies that have fully human variableregions and mouse constant regions. In another example, a targetingvector as described herein may be introduced into an engineered mouse asdescribed in any one of U.S. Pat. Nos. 9,006,511, 9,012,717, 9,029,628,9,035,128, 9,066,502, 9,150,662 and 9,163,092, incorporated herein byreference in their entireties. In some embodiments, non-human animals asdescribed herein are prepared to further comprise human immunoglobulingenes (variable and/or constant region genes). In some embodiments,non-human animals as described herein comprise an engineered Igκ lightchain locus as described herein and genetic material from a heterologousspecies (e.g., humans), wherein the genetic material encodes, in wholeor in part, one or more human heavy and/or Igκ light chain variableregions.

For example, as described herein, non-human animals comprising anengineered Igκ light chain locus as described herein may furthercomprise (e.g., via cross-breeding or multiple gene targetingstrategies) one or more modifications as described in Murphy, A. J. etal., (2014) Proc. Natl. Acad. Sci. U.S.A. 111(14):5153-8; Macdonald, L.E. et al., 2014, Proc. Natl. Acad. Sci. U.S.A. 111(14):5147-52; U.S.Pat. Nos. 8,502,018, 8,642,835, 8,697,940 and 8,791,323; all of whichare incorporated herein by reference in their entireties. In someembodiments, a rodent comprising an engineered immunoglobulin κ lightchain locus as described herein is crossed to a rodent comprising ahumanized immunoglobulin heavy chain and/or immunoglobulin κ light chainvariable region locus (see, e.g., U.S. Pat. Nos. 8,502,018, 8,642,835,8,697,940 and/or 8,791,323; incorporated herein by reference in theirentireties). In some embodiments, a rodent comprising an engineeredimmunoglobulin κ light chain locus as described herein is crossed to arodent comprising a humanized immunoglobulin heavy chain variable regionlocus (see, e.g., U.S. Pat. Nos. 8,502,018, 8,642,835, 8,697,940 and/or8,791,323; incorporated herein by reference) and an inactivatedendogenous immunoglobulin λ light chain locus (see, e.g., U.S. Pat. Nos.9,006,511, 9,012,717, 9,029,628, 9,035,128, 9,066,502, 9,150,662 and9,163,092, incorporated herein by reference in their entireties).

Although embodiments describing the construction of an engineeredimmunoglobulin κ light chain locus in a mouse (i.e., a mouse with anengineered immunoglobulin κ light chain locus characterized by thepresence of a plurality of human Vλ and Jλ gene segments operably linkedwith a mouse or human Cλ gene, which mouse or human Cλ gene is locatedin the place of a mouse Cκ gene, so that antibodies containing human Vλdomains and mouse or human Cλ domains are expressed) are extensivelydiscussed herein, other non-human animals that comprise an engineeredimmunoglobulin κ light chain locus are also provided. Such non-humananimals include any of those which can be genetically modified toexpress antibodies as described herein, including, e.g., mammals, e.g.,mouse, rat, rabbit, pig, bovine (e.g., cow, bull, buffalo), deer, sheep,goat, chicken, cat, dog, ferret, primate (e.g., marmoset, rhesusmonkey), etc. For example, for those non-human animals for whichsuitable genetically modifiable ES cells are not readily available,other methods are employed to make a non-human animal comprising thegenetic modification. Such methods include, e.g., modifying a non-EScell genome (e.g., a fibroblast or an induced pluripotent cell) andemploying somatic cell nuclear transfer (SCNT) to transfer thegenetically modified genome to a suitable cell, e.g., an enucleatedoocyte, and gestating the modified cell (e.g., the modified oocyte) in anon-human animal under suitable conditions to form an embryo.

Methods for modifying the germline genome of a non-human animal (e.g., apig, cow, rodent, chicken, etc. genome) include, e.g., employing a zincfinger nuclease (ZFN), a transcription activator-like effector nuclease(TALEN), or a Cas protein (i.e., a CRISPR/Cas system) to include anengineered immunoglobulin κ light chain locus as described herein.Guidance for methods for modifying the germline genome of a non-humananimal can be found in, e.g., U.S. patent application Ser. No.14/747,461 (filed Jun. 23, 2015), Ser. No. 14/948,221 (filed Nov. 20,2015) and Ser. No. 14/974,623 (filed Dec. 18, 2015); incorporated hereinby reference in their entireties.

In some embodiments, a non-human animal as described herein is a mammal.In some embodiments, a non-human animal as described herein is a smallmammal, e.g., of the superfamily Dipodoidea or Muroidea. In someembodiments, a genetically modified animal as described herein is arodent. In some embodiments, a rodent as described herein is selectedfrom a mouse, a rat, and a hamster. In some embodiments, a rodent asdescribed herein is selected from the superfamily Muroidea. In someembodiments, a genetically modified animal as described herein is from afamily selected from Calomyscidae (e.g., mouse-like hamsters),Cricetidae (e.g., hamster, New World rats and mice, voles), Muridae(true mice and rats, gerbils, spiny mice, crested rats), Nesomyidae(climbing mice, rock mice, with-tailed rats, Malagasy rats and mice),Platacanthomyidae (e.g., spiny dormice), and Spalacidae (e.g., molerates, bamboo rats, and zokors). In some certain embodiments, agenetically modified rodent as described herein is selected from a truemouse or rat (family Muridae), a gerbil, a spiny mouse, and a crestedrat. In some certain embodiments, a genetically modified mouse asdescribed herein is from a member of the family Muridae. In someembodiment, a non-human animal as described herein is a rodent. In somecertain embodiments, a rodent as described herein is selected from amouse and a rat. In some embodiments, a non-human animal as describedherein is a mouse.

In some embodiments, a non-human animal as described herein is a rodentthat is a mouse of a C57BL strain selected from C57BL/A, C57BL/An,C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ,C57BL/10, C57BL/10ScSn, C57BL/10Cr, and C57BL/Ola. In some certainembodiments, a mouse as described herein is a 129-strain selected fromthe group consisting of a strain that is 129P1, 129P2, 129P3, 129X1,129S1 (e.g., 129S1/SV, 129S1/SvIm), 129S2, 129S4, 129S5, 129S9/SvEvH,129/SvJae, 129S6 (129/SvEvTac), 129S7, 129S8, 129T1, 129T2 (see, e.g.,Festing et al., 1999, Mammalian Genome 10:836; Auerbach, W. et al.,2000, Biotechniques 29(5):1024-1028, 1030, 1032, each of which isincorporated herein by reference in its entirety). In some certainembodiments, a genetically modified mouse as described herein is a mixof an aforementioned 129 strain and an aforementioned C57BL/6 strain. Insome certain embodiments, a mouse as described herein is a mix ofaforementioned 129 strains, or a mix of aforementioned BL/6 strains. Insome certain embodiments, a 129 strain of the mix as described herein isa 129S6 (129/SvEvTac) strain. In some embodiments, a mouse as describedherein is a BALB strain, e.g., BALB/c strain. In some embodiments, amouse as described herein is a mix of a BALB strain and anotheraforementioned strain.

In some embodiments, a non-human animal as described herein is a rat. Insome certain embodiments, a rat as described herein is selected from aWistar rat, an LEA strain, a Sprague Dawley strain, a Fischer strain,F344, F6, and Dark Agouti. In some certain embodiments, a rat strain asdescribed herein is a mix of two or more strains selected from the groupconsisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and DarkAgouti.

A rat pluripotent and/or totipotent cell can be from any rat strain,including, for example, an ACI rat strain (an inbred strain originallyderived from August and Copenhagen strains), a Dark Agouti (DA) ratstrain, a Wistar rat strain, a LEA rat strain, a Sprague Dawley (SD) ratstrain, or a Fischer rat strain such as Fisher F344 or Fisher F6. Ratpluripotent and/or totipotent cells can also be obtained from a strainderived from a mix of two or more strains recited above. For example,the rat pluripotent and/or totipotent cell can be from a DA strain or anACI strain. The ACI rat strain is characterized as having black agouti,with white belly and feet and an RTlav1 haplotype. Such strains areavailable from a variety of sources including Harlan Laboratories. Anexample of a rat ES cell line from an ACI rat is an ACI.G1 rat ES cell.The DA rat strain is characterized as having an agouti coat and anRTlav1 haplotype. Such rats are available from a variety of sourcesincluding Charles River and Harlan Laboratories. Examples of a rat EScell line from a DA rat are the DA.2B rat ES cell line and the DA.2C ratES cell line. In some embodiments, the rat pluripotent and/or totipotentcells are from an inbred rat strain (see, e.g., U.S. Patent ApplicationPublication No. 2014-0235933 A1, published Aug. 21, 2014, incorporatedherein by reference in its entirety). Guidance for making modificationsin a rat genome (e.g., in a rat ES cell) using methods and/or constructsas described herein can be found in, e.g., in U.S. Patent ApplicationPublication Nos. 2014-0310828 and 2017-0204430; both of which areincorporated herein by reference in their entireties.

Specific Exemplary Embodiments—Immunoglobulin Heavy Chain Loci

In some embodiments, provided non-human animals comprise an engineeredimmunoglobulin κ light chain locus as described herein and furthercomprise engineered IgH loci (or alleles) characterized by the presenceof a plurality of human V_(H), D_(H) and J_(H) gene segments arranged ingermline configuration and operably linked to non-human immunoglobulinheavy chain constant region genes, enhancers and regulatory regions. Insome embodiments, an engineered immunoglobulin heavy chain locus (orallele) as described herein comprises one or more human V_(H) genesegments, one or more human D_(H) gene segments and one or more humanJ_(H) gene segments operably linked to a non-human immunoglobulin heavychain constant region. In some certain embodiments, an engineeredimmunoglobulin heavy chain locus (or allele) comprises at least humanV_(H) gene segments V_(H)3-74, V_(H)3-73, V_(H)3-72, V_(H)2-70,V_(H)1-69, V_(H)3-66, V_(H)3-64, V_(H)4-61, V_(H)4-59, V_(H)1-58,V_(H)3-53, V_(H)5-51, V_(H)3-49, V_(H)3-48, V_(H)1-46, V_(H)1-45,V_(H)3-43, V_(H)4-39, V_(H)4-34, V_(H)3-33, V_(H)4-31, V_(H)3-30,V_(H)4-28, V_(H)2-26, V_(H)1-24, V_(H)3-23, V_(H)3-21, V_(H)3-20,V_(H)1-18, V_(H)3-15, V_(H)3-13, V_(H)3-11, V_(H)3-9, V_(H)1-8,V_(H)3-7, V_(H)2-5, V_(H)7-4-1, V_(H)4-4, V_(H)1-3, V_(H)1-2, V_(H)6-1,or any combination thereof. In some certain embodiments, an engineeredIgH locus (or allele) comprises at least human D_(H) gene segmentsD_(H)1-1, D_(H)2-2, D_(H)3-3, D_(H)4-4, D_(H)5-5, D_(H)6-6, D_(H)1-7,D_(H)2-8, D_(H)3-9, D_(H)3-10, D_(H)5-12, D_(H)6-13, D_(H)2-15,D_(H)3-16, D_(H)4-17, D_(H)6-19, D_(H)1-20, D_(H)2-21, D_(H)3-22,D_(H)6-25, D_(H)1-26, D_(H)7-27, or any combination thereof. In somecertain embodiments, an engineered immunoglobulin heavy chain locus (orallele) comprises at least human J_(H) gene segments J_(H)1, J_(H)2,J_(H)3, J_(H)4, J_(H)5, J_(H)6, or any combination thereof.

The present disclosure recognizes that a non-human animal as describedherein will utilize human heavy chain variable region gene segmentscomprised in its genome in its antibody selection and generationmechanisms (e.g., recombination and somatic hypermutation). As such, invarious embodiments, human immunoglobulin heavy chain variable domainsgenerated by non-human animals described herein are encoded by the humanheavy chain variable region gene segments included in their genome orsomatically hypermutated variants thereof.

In some embodiments, a non-human animal is provided whose genomecomprises an engineered immunoglobulin κ light chain locus, where thenon-human animal includes a B cell that includes a human heavy variableregion sequence, a human λ light chain variable region sequence, and/ora human κ light chain variable region sequence that is somaticallyhypermutated. In some embodiments, a human heavy variable regionsequence, a human λ light chain variable region sequence, and/or a humanκ light chain variable region sequence present in a B cell of a mouse ofthe present disclosure has 1, 2, 3, 4, 5, or more somatichypermutations. Those skilled in the art are aware of methods foridentifying source gene segments in a mature antibody sequence. Forexample, various tools are available to aid in this analysis, such as,for example, DNAPLOT, IMGT/V-QUEST, JOINSOLVER, SoDA, and Ab-origin.

In some embodiments, a non-human immunoglobulin heavy chain constantregion includes one or more non-human immunoglobulin heavy chainconstant region genes such as, for example, immunoglobulin M (IgM),immunoglobulin D (IgD), immunoglobulin G (IgG), immunoglobulin E (IgE)and immunoglobulin A (IgA). In some certain embodiments, a non-humanimmunoglobulin heavy chain constant region includes a rodent IgM, rodentIgD, rodent IgG3, rodent IgG1, rodent IgG2b, rodent IgG2a, rodent IgEand rodent IgA constant region genes. In some embodiments, said humanV_(H), D_(H) and J_(H) gene segments are operably linked to one or morenon-human immunoglobulin heavy chain enhancers (i.e., enhancer sequencesor enhancer regions). In some embodiments, said human V_(H), D_(H) andJ_(H) gene segments are operably linked to one or more non-humanimmunoglobulin heavy chain regulatory regions (or regulatory sequences).In some embodiments, said human V_(H), D_(H) and J_(H) gene segments areoperably linked to one or more non-human immunoglobulin heavy chainenhancers (or enhancer sequence) and one or more non-humanimmunoglobulin heavy chain regulatory regions (or regulatory sequence).

In some embodiments, an engineered immunoglobulin heavy chain locus asdescribed herein does not contain an endogenous Adam6 gene. In someembodiments, an engineered immunoglobulin heavy chain locus as describedherein does not contain an endogenous Adam6 gene (or Adam6-encodingsequence) in the same germline genomic position as found in a germlinegenome of a wild-type non-human animal of the same species. In someembodiments, an engineered immunoglobulin heavy chain locus as describedherein does not contain a human Adam6 pseudogene. In some embodiments,an engineered immunoglobulin heavy chain locus as described hereincomprises insertion of at least one nucleotide sequence that encodes oneor more non-human (e.g., rodent) Adam6 polypeptides, functionalorthologs, functional homologs, or functional fragments thereof. In someembodiments, said insertion may be outside of an engineeredimmunoglobulin heavy chain locus as described herein (e.g., but notlimited to, upstream of a 5′ most V_(H) gene segment), within anengineered immunoglobulin heavy chain locus or elsewhere in the germlinegenome of a non-human animal (e.g., but not limited to, a randomlyintroduced non-human Adam6-encoding sequence), cell or tissue.

In various embodiments, a provided non-human animal, non-human cell ornon-human tissue as described herein does not detectably express, inwhole or in part, an endogenous non-human V_(H) region in an antibodymolecule. In various embodiments, a provided non-human animal, non-humancell or non-human tissue as described herein does not contain (or lacks,or contains a deletion of) one or more nucleotide sequences that encode,in whole or in part, an endogenous non-human V_(H) region (e.g., V_(H),D_(H) and/or J_(H)) in an antibody molecule. In various embodiments, aprovided non-human animal, non-human cell or non-human tissue asdescribed herein has a germline genome that includes a deletion ofendogenous non-human V_(H), D_(H) and J_(H) gene segments, in whole orin part. In various embodiments, a provided non-human animal is fertile.

Guidance for the creation of targeting vectors, non-human cells andanimals harboring such engineered immunoglobulin heavy chain loci (oralleles) can be found in U.S. Pat. Nos. 8,502,018, 8,642,835, 8,697,940and 8,791,323, each of which is incorporated herein by reference in itsentirety. Persons skilled in the art are aware of a variety oftechnologies, known in the art, for accomplishing such geneticengineering and/or manipulation of non-human (e.g., mammalian) genomesor for otherwise preparing, providing, or manufacturing such sequencesfor introducing into the germline genome of non-human animals.

Specific Exemplary Embodiments—Immunoglobulin κ Light Chain Loci

In some embodiments, provided non-human animals comprise an engineeredimmunoglobulin κ light chain locus characterized by the presence of aplurality of human Vλ and Jλ gene segments arranged in germlineconfiguration (i.e., not rearranged and associated with recombinationsignal sequences) and inserted upstream of, and operably linked to, anon-human or human Cλ gene, which non-human or human Cλ gene is insertedin the place of a non-human Cκ gene. As described herein, suchengineered immunoglobulin κ light chain locus further includes non-humanimmunoglobulin κ light chain enhancer regions (or enhancer sequences).In some embodiments, an engineered immunoglobulin κ light chain locuscomprises one or more human Vλ gene segments and one or more human Jλgene segments operably linked to a non-human or human Cλ gene. In somecertain embodiments, an engineered immunoglobulin κ light chain locus(or allele) comprises human Vλ gene segments that appear in at leastcluster A of a human immunoglobulin λ light chain locus; in someembodiments, cluster A and cluster B of a human immunoglobulin λ lightchain locus; in some certain embodiments, cluster A, cluster B andcluster C of a human immunoglobulin λ light chain locus. In some certainembodiments, an engineered immunoglobulin κ light chain locus (orallele) comprises at least human Vλ gene segments Vλ4-69, Vλ8-61,Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45,Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23,Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9,Vλ2-8, Vλ4-3, Vλ3-1 or any combination thereof. In some certainembodiments, an engineered Igκ light chain locus (or allele) comprisesat least human Jλ gene segments Jλ1, Jλ2, Jλ3, Jλ6 Jλ7, or anycombination thereof.

The present disclosure recognizes that a non-human animal as describedherein will utilize human λ light chain variable region gene segmentsincluded in its genome in its antibody selection and generationmechanisms (e.g., recombination and somatic hypermutation). As such, invarious embodiments, human immunoglobulin λ light chain variable domainsgenerated by non-human animals described herein are encoded by the humanλ light chain variable region gene segments included in their genome orsomatically hypermutated variants thereof.

In some embodiments, a non-human animal is provided whose genomecomprises an engineered immunoglobulin κ light chain locus, where thenon-human animal includes a B cell that includes a human heavy variableregion sequence, a human λ light chain variable region sequence, and/ora human κ light chain variable region sequence that is somaticallyhypermutated. In some embodiments, a human heavy variable regionsequence, a human λ light chain variable region sequence, and/or a humanκ light chain variable region sequence present in a B cell of a mouse ofthe present disclosure has 1, 2, 3, 4, 5, or more somatichypermutations. Those skilled in the art are aware of methods foridentifying source gene segments in a mature antibody sequence. Forexample, various tools are available to aid in this analysis, such as,for example, DNAPLOT, IMGT/V-QUEST, JOINSOLVER, SoDA, and Ab-origin.

In many embodiments, an engineered immunoglobulin κ light chain locus(or allele) contains the same non-human immunoglobulin κ light chainenhancer regions (or enhancer sequences) that appear in a wild-typeimmunoglobulin κ light chain locus (or allele). In some embodiments, anengineered immunoglobulin κ light chain locus (or allele) containsnon-human immunoglobulin κ light chain enhancer regions (or enhancersequences) that appear in a wild-type immunoglobulin κ light chain locus(or allele) of a different species (e.g., a different rodent species).

In some embodiments, said human Vλ and Jλ gene segments are operablylinked to one or more non-human immunoglobulin κ light chain enhancers(i.e., enhancer sequences or enhancer regions). In some certainembodiments, said human Vλ and Jλ gene segments are operably linked to amurine immunoglobulin κ light chain intronic enhancer region (Igκ Ei orEiκ). In some certain embodiments, said human Vλ and Jλ gene segmentsare operably linked to a murine immunoglobulin κ light chain 3′ enhancerregion (Igκ 3′E or 3′Eκ). In some certain embodiments, said human Vλ andJλ gene segments are operably linked to a murine Eiκ and operably linkedto a murine 3′Eκ.

In some embodiments, an engineered immunoglobulin κ light chain locus(or allele) as described herein does not contain (i.e., lacks) a humanVpreB gene (or human VpreB gene-encoding sequence).

In some embodiments, a non-human Cλ gene of an engineered immunoglobulinκ light chain locus (or allele) includes a rodent Cλ gene such as, forexample, a mouse Cλ gene or a rat Cλ gene. In some certain embodiments,a non-human Cλ gene of an engineered Igκ light chain locus (or allele)is or comprises a mouse Cλ gene from a genetic background that includesa 129 strain, a BALB/c strain, a C57BL/6 strain, a mixed 129xC57BL/6strain or combinations thereof.

In some embodiments, a non-human Cλ gene of an engineered Igκ lightchain locus (or allele) as described herein comprises a sequence that isat least 80%, at least 85%, at least 90%, at least 95%, or at least 98%identical to SEQ ID NO: 1 (mouse Cλ1), SEQ ID NO:3 (mouse Cλ2) or SEQ IDNO:5 (mouse Cλ3). In some embodiments, a non-human Cλ gene of anengineered Igκ light chain locus (or allele) as described hereincomprises a sequence that is substantially identical or identical to SEQID NO: 1 (mouse Cλ1), SEQ ID NO:3 (mouse Cλ2) or SEQ ID NO:5 (mouseCλ3). In some embodiments, a non-human Cλ gene of an engineered Igκlight chain locus (or allele) as described herein is or comprises thesequence of a mouse Cλ1 gene.

In some embodiments, a non-human Cλ domain encoded by a sequencepositioned at an engineered Igκ light chain locus (or allele) asdescribed herein comprises a sequence that is at least 80%, at least85%, at least 90%, at least 95%, or at least 98% identical to SEQ IDNO:2 (mouse Cλ1), SEQ ID NO:4 (mouse Cλ2) or SEQ ID NO:6 (mouse Cλ3). Insome embodiments, a non-human Cλ domain encoded by a sequence positionedat an engineered Igκ light chain locus (or allele) as described hereincomprises a sequence that is substantially identical or identical to SEQID NO:2 (mouse Cλ1), SEQ ID NO:4 (mouse Cλ2) or SEQ ID NO:6 (mouse Cλ3).In some embodiments, a non-human Cλ gene encoded by a sequencepositioned at an engineered Igκ light chain locus (or allele) asdescribed herein is or comprises a mouse Cλ1 domain polypeptide.

In some embodiments, a non-human Cλ gene of an engineered Igκ lightchain locus (or allele) as described herein comprises a sequence that isat least 80%, at least 85%, at least 90%, at least 95%, or at least 98%identical to SEQ ID NO:7 (rat Cλ1), SEQ ID NO:9 (rat Cλ2), SEQ ID NO: 11(rat Cλ3) or SEQ ID NO: 13 (rat Cλ4). In some certain embodiments, anon-human Cλ gene of an engineered Igκ light chain locus (or allele) asdescribed herein comprises a sequence that is substantially identical oridentical to SEQ ID NO:7 (rat Cλ1), SEQ ID NO:9 (rat Cλ2), SEQ ID NO: 11(rat Cλ3) or SEQ ID NO: 13 (rat Cλ4). In some certain embodiments, anon-human Cλ gene of an engineered Igκ light chain locus (or allele) asdescribed herein is or comprises the sequence of a rat Cλ1 gene.

In some embodiments, a non-human Cλ domain encoded by a sequencepositioned at an engineered Igκ light chain locus (or allele) asdescribed herein comprises a sequence that is at least 80%, at least85%, at least 90%, at least 95%, or at least 98% identical to SEQ IDNO:8 (rat Cλ1), SEQ ID NO:10 (rat Cλ2), SEQ ID NO:12 (rat Cλ3) or SEQ IDNO:14 (rat Cλ4). In some embodiments, a non-human Cλ domain encoded by asequence positioned at an engineered Igκ light chain locus (or allele)as described herein comprises a sequence that is substantially identicalor identical to SEQ ID NO:8 (rat Cλ1), SEQ ID NO: 10 (rat Cλ2), SEQ IDNO: 12 (rat Cλ3) or SEQ ID NO:14 (rat Cλ4). In some embodiments, anon-human Cλ domain encoded by a sequence positioned at an engineeredIgκ light chain locus (or allele) as described herein is or comprises arat Cλ1 domain polypeptide.

In some embodiments, a human Cλ gene of an engineered Igκ light chainlocus (or allele) includes a human Cλ gene such as, for example, a humanCλ1 gene, a human Cλ2 gene, a human Cλ3 gene, a human Cλ6 gene or ahuman Cλ7 gene. In some certain embodiments, a human Cλ gene of anengineered Igκ light chain locus (or allele) is or comprises a human Cλ2gene.

In some embodiments, a human Cλ gene of an engineered Igκ light chainlocus (or allele) as described herein comprises a sequence that is atleast 80%, at least 85%, at least 90%, at least 95%, or at least 98%identical to SEQ ID NO:15 (human Cλ1), SEQ ID NO:17 (human Cλ2), SEQ IDNO: 19 (human Cλ3), SEQ ID NO:21 (human Cλ6) or SEQ ID NO:23 (humanCλ7). In some embodiments, a human Cλ gene of an engineered Igκ lightchain locus (or allele) as described herein comprises a sequence that issubstantially identical or identical to SEQ ID NO:15 (human Cλ1), SEQ IDNO: 17 (human Cλ2), SEQ ID NO: 19 (human Cλ3), SEQ ID NO:21 (human Cλ6)or SEQ ID NO:23 (human Cλ7). In some embodiments, a human Cλ gene of anengineered Igκ light chain locus (or allele) as described herein is orcomprises the sequence of a human Cλ2 gene.

In some embodiments, a human Cλ domain encoded by a sequence positionedat an engineered Igκ light chain locus (or allele) as described hereincomprises a sequence that is at least 80%, at least 85%, at least 90%,at least 95%, or at least 98% identical to SEQ ID NO: 16 (human Cλ1),SEQ ID NO:18 (human Cλ2), SEQ ID NO:20 (human Cλ3), SEQ ID NO:22 (humanCλ6) or SEQ ID NO:24 (human Cλ7). In some embodiments, a human Cλ domainencoded by a sequence positioned at an engineered Igκ light chain locus(or allele) as described herein comprises a sequence that issubstantially identical or identical to SEQ ID NO: 16 (human Cλ1), SEQID NO: 18 (human Cλ2), SEQ ID NO:20 (human Cλ3), SEQ ID NO:22 (humanCλ6) or SEQ ID NO:24 (human Cλ7). In some embodiments, a human Cλ domainencoded by a sequence positioned at an engineered Igκ light chain locus(or allele) as described herein is or comprises a human Cλ2 domainpolypeptide.

Among other things, the present disclosure demonstrates that thepresence of human Vλ and Jλ gene segments at Igκ light chain loci (oralleles) increases the diversity of the light chain repertoire of aprovided non-human animal as compared to the diversity of the lightchains in the expressed antibody repertoire of a non-human animal thatdoes not comprise such engineered Igκ light chain loci.

Specific Exemplary Embodiments—Immunoglobulin λ Light Chain Loci

In some embodiments, provided non-human animals comprise an engineeredimmunoglobulin κ light chain locus as described herein and furthercomprise wild-type or inactivated immunoglobulin λ light chain loci (oralleles).

In some embodiments, provided non-human animals, non-human cells and/ornon-human tissues as described herein comprise a deletion, in whole orin part, of an endogenous immunoglobulin λ light chain locus. In someembodiments, provided non-human animals, non-human cells and/ornon-human tissues as described herein comprise an insertion within anendogenous immunoglobulin λ light chain locus, wherein said insertionrenders the endogenous immunoglobulin λ light chain locusnon-functional. In some embodiments, provided non-human animals,non-human cells and/or non-human tissues as described herein comprise adeletion of one or more gene segments of an endogenous immunoglobulin λlight chain locus such that the endogenous immunoglobulin λ light chainlocus is unable to recombine and/or express a functional light chain ofan antibody.

In some embodiments, a provided non-human animal, non-human cell ornon-human tissue as described herein does not detectably express, inwhole or in part, an endogenous non-human Vλ region in an antibodymolecule. In some embodiments, a provided non-human animal, non-humancell or non-human tissue as described herein does not contain (or lacks,or contains a deletion of) one or more nucleotide sequences that encode,in whole or in part, an endogenous non-human Vλ region in an antibodymolecule. In some embodiments, a provided non-human animal, non-humancell or non-human tissue as described herein has a germline genome thatincludes a deletion of endogenous non-human Vλ and Jλ gene segments, inwhole or in part. In some embodiments, a provided non-human animal,non-human cell or non-human tissue as described herein as a germlinegenome that includes a deletion of endogenous non-human Vλ, Jλ and Cλgene segments, in whole or in part.

Guidance for the creation of targeting vectors, non-human cells andanimals harboring inactivated Igλ light chain loci (or alleles) can befound in, e.g., U.S. Pat. Nos. 9,006,511, 9,012,717, 9,029,628,9,035,128, 9,066,502, 9,150,662 and 9,163,092, which are incorporatedherein by reference in their entireties. Those skilled in the art areaware of a variety of technologies, known in the art, for accomplishinggenetic inactivation of specific loci and/or manipulation of non-human(e.g., mammalian) genomes or for otherwise preparing, providing, ormanufacturing such genetic inactivation (e.g., gene deletions) forintroducing into the germline genome of non-human animals.

Specific Exemplary Embodiments—Combinations of Immunoglobulin Loci

In some embodiments, provided non-human animals comprise an engineeredimmunoglobulin κ light chain locus as described herein and furthercomprise one or more additional human or humanized genes (e.g., viacross-breeding or multiple gene targeting strategies). Such non-humananimals may be prepared as described above, or using methods known inthe art, to achieve a desired engineered genotype depending on theintended use of the non-human animal. Genetic material of suchadditional human or humanized genes may be introduced through thefurther alteration of the genome of cells (e.g., embryonic stem cells)having the genetic modifications as described above or through breedingtechniques known in the art with other genetically modified strains asdesired.

In some embodiments, provided non-human animals are prepared to furthercomprise a human or humanized immunoglobulin heavy chain locus (e.g.,including but not limited to, a plurality of human V_(H), D_(H) andJ_(H) gene segments operably linked to one or more rodent immunoglobulinheavy chain constant region genes at an endogenous immunoglobulin heavychain locus). In some embodiments, provided non-human animals areheterozygous for an engineered immunoglobulin κ light chain locus asdescribed herein and heterozygous for a human or humanizedimmunoglobulin heavy chain locus. In some embodiments, providednon-human animals are homozygous for an engineered immunoglobulin κlight chain locus as described herein and homozygous for a human orhumanized immunoglobulin heavy chain locus.

In some embodiments, provided non-human animals are prepared to furthercomprise a human or humanized immunoglobulin heavy chain locus (e.g.,including but not limited to, a plurality of human V_(H), D_(H) andJ_(H) gene segments operably linked to one or more rodent immunoglobulinheavy chain constant region genes at an endogenous immunoglobulin heavychain locus) and a humanized immunoglobulin κ light chain locus (e.g.,including but not limited to, a plurality of human Vκ and Jκ genesegments operably linked to a rodent immunoglobulin κ light chainconstant region gene at an endogenous immunoglobulin κ light chainlocus. In some embodiments, provided non-human animals are heterozygousfor a human or humanized immunoglobulin heavy chain locus and furthercomprise one endogenous immunoglobulin κ light chain locus that containsa plurality of human Vλ and Jλ gene segments operably linked to a rodentimmunoglobulin λ light chain constant region gene (i.e., an engineeredIgκ light chain locus as described herein), and another endogenousimmunoglobulin κ light chain locus having a plurality of human Vκ and Jκgene segments operably linked to a rodent immunoglobulin κ light chainconstant region gene. In some embodiments, provided non-human animalsare homozygous for a human or humanized immunoglobulin heavy chain locusand further comprise an endogenous immunoglobulin κ light chain locusthat contains a plurality of human Vλ and Jλ gene segments operablylinked to a rodent immunoglobulin λ light chain constant region gene(i.e., an engineered Igκ light chain locus as described herein), andanother endogenous immunoglobulin κ light chain locus having a pluralityof human Vκ and Jκ gene segments operably linked to a rodentimmunoglobulin κ light chain constant region gene.

In some embodiments, provided non-human animals have a genome comprising(a) a homozygous or heterozygous human or humanized immunoglobulin heavychain locus comprising human V_(H), D_(H) and J_(H) gene segmentsoperably linked to one or more endogenous non-human immunoglobulin heavychain constant regions such that the non-human animal expresses animmunoglobulin heavy chain that comprises a human V_(H) domain sequencefused with a non-human C_(H) domain sequence; (b) a first immunoglobulinκ light chain locus comprising human Vλ and Jλ gene segments operablylinked to a non-human immunoglobulin Cλ gene such that the non-humananimal expresses an immunoglobulin light chain that comprises a human Vλdomain sequence fused with a non-human Cλ domain sequence; and (c) asecond immunoglobulin κ light chain locus comprising human Vx and Jκgene segments operably linked to an endogenous non-human Cκ gene suchthat the non-human animal expresses an immunoglobulin light chain thatcomprises a human Vκ domain sequence fused with a mouse Cκ domainsequence.

In some embodiments, provided non-human animals have a genome comprising(a) a homozygous or heterozygous human or humanized immunoglobulin heavychain locus comprising human V_(H), D_(H) and J_(H) gene segmentsoperably linked to one or more endogenous non-human immunoglobulin heavychain constant regions such that the non-human animal expresses animmunoglobulin heavy chain that comprises a human V_(H) domain sequencefused with a non-human C_(H) domain sequence; (b) a first immunoglobulinκ light chain locus comprising human Vλ and Jλ gene segments operablylinked to a non-human immunoglobulin Cλ gene such that the non-humananimal expresses an immunoglobulin light chain that comprises a human Vλdomain sequence fused with a non-human Cλ domain sequence; (c) a secondimmunoglobulin κ light chain locus comprising human Vx and Jκ genesegments operably linked to an endogenous non-human Cκ gene such thatthe non-human animal expresses an immunoglobulin light chain thatcomprises a human Vκ domain sequence fused with a mouse Cκ domainsequence; and (d) a homozygous or heterozygous functionally inactivatedor deleted, in whole or in part, endogenous immunoglobulin λ light chainlocus.

For example, as described herein, non-human animals comprising anengineered immunoglobulin κ light chain locus as described herein mayfurther comprise (e.g., via cross-breeding or multiple gene targetingstrategies) one or more modifications as described in U.S. Pat. Nos.8,642,835, 8,697,940, 9,006,511, 9,035,128, 9,066,502, 9,150,662 and9,163,092; each of which is incorporated by reference in its entirety.In some embodiments, provided non-human animals further comprise ahumanized immunoglobulin heavy chain locus (e.g., an immunoglobulinheavy chain locus comprising human V_(H), D_(H) and J_(H) gene segmentsoperably linked to one or more non-human immunoglobulin heavy chainconstant region genes). In some embodiments, provided non-human animalsfurther comprise a humanized immunoglobulin heavy chain locus and anon-functional endogenous immunoglobulin λ light chain locus (e.g.,deleted in whole or in part, or otherwise rendered non-functional).

In some embodiments, provided non-human animals comprise animmunoglobulin κ light chain locus having human Vλ and Jλ gene segmentsoperably linked to a human or non-human Cλ gene positioned in the placeof a non-human Cκ gene and a second immunoglobulin κ light chain locuscomprising human Vκ and Jκ gene segments operably linked to anendogenous non-human Cκ gene. In such embodiments, provided non-humananimals are referred to as hemizygous for an engineered immunoglobulin κlight chain locus. In some embodiment, said hemizygous non-human animalsprovided herein further comprise a humanized immunoglobulin heavy chainlocus. In some embodiments, said hemizygous non-human animals providedherein further comprise a humanized immunoglobulin heavy chain locus anda non-functional endogenous immunoglobulin λ light chain locus.

Methods of Using Provided Non-Human Animals, Cells or Tissues

Non-human animals as described herein can be used as a platform for thedevelopment of antibodies. In particular, the non-human animalsdescribed herein represent a particularly advantageous platform for thegeneration and identification of human lambda light chain variabledomains and antibodies that include such human lambda light chainvariable domains.

Accordingly, the present disclosure provides that non-human animalsdescribed herein can be used in methods of making antibodies. Antibodiesmade in accordance with the present disclosure can include, for example,human antibodies, chimeric antibodies, reverse chimeric antibodies,fragments of any of these antibodies, or combinations thereof.

In some embodiments, non-human animals as described herein may beemployed for making a human antibody (e.g., a fully human antibody),which human antibody comprises variable regions derived from nucleicacid sequences encoded by genetic material of a cell of a non-humananimal as described herein. In some embodiments, a non-human animal asdescribed herein is immunized with an antigen of interest underconditions and for a time sufficient that the non-human animal developsan immune response to said antigen of interest. Antibodies and/orantibody sequences (i.e., sequences that encode for part of an antibody,e.g., a variable region sequence) are isolated and/or identified fromthe non-human animal (or one or more cells, for example, one or more Bcells) so immunized and characterized using various assays measuring,for example, affinity, specificity, epitope mapping, ability forblocking ligand-receptor interaction, inhibition receptor activation,etc. In various embodiments, antibodies produced by non-human animals asdescribed herein comprise one or more human variable regions that arederived from one or more human variable region nucleotide sequencesisolated from the non-human animal. In some embodiments, anti-drugantibodies (e.g., anti-idiotype antibody) may be raised in non-humananimals as described herein. In various embodiments, antibodies producedby non-human animals as described herein are reverse chimeric antibodiesthat include a human light chain variable domain and a non-human (e.g.,rodent) light chain constant domain and/or a human heavy chain variabledomain and a non-human (e.g. rodent) heavy chain constant domain.

In various embodiments, antibodies produced by non-human animals includeheavy and light chains having a human variable domain and a non-humanconstant domain. In some embodiments, antibodies produced by non-humananimals as described herein are reverse chimeric antibodies that includea human light chain variable domain and a non-human (e.g. rodent) lightchain constant domain. In some embodiments, antibodies produced bynon-human animals as described herein are reverse chimeric antibodiesthat include a human heavy chain variable domain and a non-human (e.g.rodent) heavy chain constant domain.

In some embodiments, provided methods include immunizing a non-humananimal as described herein with an antigen of interest. In someembodiments, provided methods include identifying a lymphocyte (e.g., aclonally selected lymphocyte) from said non-human animal, where thelymphocyte expresses an antibody that binds (e.g., specifically binds)the antigen of interest. In some embodiments, a lymphocyte is a B cell.In some embodiments, a human heavy chain variable region sequence, ahuman lambda light chain variable region sequence, and/or a human kappalight chain variable region sequence is obtained from the lymphocyte(e.g., B cell) and/or identified (e.g., genotyped, e.g., sequenced). Insome embodiments, an amino acid sequence of a human heavy chain variabledomain, a human lambda light chain variable domain, and/or a human kappalight chain variable domain is obtained from the lymphocyte (e.g., Bcell) and/or identified (e.g., sequenced). In some embodiments, a humanheavy chain variable region sequence, a human lambda light chainvariable region sequence, and/or a human kappa light chain variableregion sequence from a B cell of a non-human animal is expressed in ahost cell. In some embodiments, a variant of a human heavy chainvariable region sequence, a human lambda light chain variable regionsequence, and/or a human kappa light chain variable region sequence froma B cell of a non-human animal is expressed in a host cell. In someembodiments, a variant includes one or more mutations. In someembodiments, one or more mutations can improve a pharmacokinetic and/ora pharmacodynamic property of an antibody including a variant. In someembodiments, one or more mutations can improve the specificity, theaffinity, and/or the immunogenicity of an antibody including a variant.

In some embodiments, methods of making a human antibody includeidentifying a nucleotide sequence encoding a human immunoglobulin heavychain variable domain and/or a human immunoglobulin light chain variabledomain from a non-human animal described herein; and (i) joining orligating the nucleotide sequence encoding the human immunoglobulin heavychain variable domain to a nucleotide sequence encoding a humanimmunoglobulin heavy chain constant domain, thereby forming a humanimmunoglobulin heavy chain sequence encoding a fully humanimmunoglobulin heavy chain, (ii) joining or ligating the nucleotidesequence encoding the human immunoglobulin λ light chain variable domainto a nucleotide sequence encoding a human immunoglobulin λ light chainconstant domain, thereby forming a human immunoglobulin λ light chainsequence encoding a fully human immunoglobulin λ light chain, and/or(iii) joining or ligating the nucleotide sequence encoding the humanimmunoglobulin κ light chain variable domain to a nucleotide sequenceencoding a human immunoglobulin κ light chain constant domain, therebyforming a human immunoglobulin κ light chain sequence encoding a fullyhuman immunoglobulin κ light chain. In certain embodiments, a humanimmunoglobulin heavy chain sequence, and (i) a human immunoglobulin λlight chain sequence, or (ii) a human immunoglobulin κ light chainsequence are expressed in a cell (e.g., a host cell, a mammalian cell)so that fully human immunoglobulin heavy chains and (i) fully humanimmunoglobulin λ light chains or (ii) fully human immunoglobulin κ lightchains are expressed and form human antibodies. In some embodiments,human antibodies are isolated from the cell or culture media includingthe cell.

Non-human animals as described herein may be employed for identifying anucleotide or nucleic acid sequence encoding a human variable domaingenerated by a non-human animal described herein, e.g., as part of anantibody against an epitope or antigen.

Non-human animals as described herein may be employed for identifying anamino acid sequence of a human variable domain generated by a non-humananimal described herein, e.g., as part of an antibody against an epitopeor antigen.

Non-human animals as described herein provide an improved in vivo systemand source of biological materials (e.g., cells, nucleotides,polypeptides, protein complexes) for producing human antibodies that areuseful for a variety of assays. In various embodiments, non-humananimals as described herein are used to develop therapeutics that targeta polypeptide of interested (e.g., a transmembrane or secretedpolypeptide) and/or modulate one or more activities associated with saidpolypeptide of interest and/or modulate interactions of said polypeptideof interest with other binding partners (e.g., a ligand or receptorpolypeptide). For example, in various embodiments, non-human animals asdescribed herein are used to develop therapeutics that target one ormore receptor polypeptides, modulate receptor polypeptide activityand/or modulate receptor polypeptide interactions with other bindingpartners. In various embodiments, non-human animals as described hereinare used to identify, screen and/or develop candidate therapeutics(e.g., antibodies, siRNA, etc.) that bind one or more polypeptides ofinterest. In various embodiments, non-human animals as described hereinare used to screen and develop candidate therapeutics (e.g., antibodies,siRNA, etc.) that block activity of one or more polypeptides of interestor that block the activity of one or more receptor polypeptides ofinterest. In various embodiments, non-human animals as described hereinare used to determine the binding profile of antagonists and/or agonistsof one or more polypeptides of interest. In some embodiments, non-humananimals as described herein are used to determine the epitope orepitopes of one or more candidate therapeutic antibodies that bind oneor more polypeptides of interest.

In various embodiments, non-human animals as described herein are usedto determine the pharmacokinetic profiles of one or more human antibodycandidates. In various embodiments, one or more non-human animals asdescribed herein and one or more control or reference non-human animalsare each exposed to one or more human antibody candidates at variousdoses (e.g., 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 1mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/mg, 7.5 mg/kg, 10 mg/kg, 15mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, or 50 mg/kg or more).Candidate therapeutic antibodies may be dosed via any desired route ofadministration including parenteral and non-parenteral routes ofadministration. Parenteral routes include, e.g., intravenous,intraarterial, intraportal, intramuscular, subcutaneous,intraperitoneal, intraspinal, intrathecal, intracerebroventricular,intracranial, intrapleural or other routes of injection. Non-parenteralroutes include, e.g., oral, nasal, transdermal, pulmonary, rectal,buccal, vaginal, ocular. Administration may also be by continuousinfusion, local administration, sustained release from implants (gels,membranes or the like), and/or intravenous injection. Blood is isolatedfrom non-human animals (humanized and control) at various time points(e.g., 0 hr, 6 hr, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7days, 8 days, 9 days, 10 days, 11 days, or up to 30 or more days).Various assays may be performed to determine the pharmacokineticprofiles of administered candidate therapeutic antibodies using samplesobtained from non-human animals as described herein including, but notlimited to, total IgG, anti-therapeutic antibody response,agglutination, etc.

In various embodiments, non-human animals as described herein are usedto measure the therapeutic effect of blocking or modulating the activityof a polypeptide of interest and the effect on gene expression as aresult of cellular changes or, in the context of a receptor polypeptide,the density of a receptor polypeptide on the surface of cells in thenon-human animals. In various embodiments, a non-human animal asdescribed herein or cells isolated therefrom are exposed to a candidatetherapeutic that binds a polypeptide of interest and, after a subsequentperiod of time, analyzed for effects on specific cellular processes thatare associated with said polypeptide of interest, for example,ligand-receptor interactions or signal transduction.

Non-human animals as described herein express human antibody variableregions, thus cells, cell lines, and cell cultures can be generated toserve as a source of human antibody variable regions for use in bindingand functional assays, e.g., to assay for binding or function of anantagonist or agonist, particularly where the antagonist or agonist isspecific for a human antigen of interest or specific for an epitope thatfunctions in ligand-receptor interaction (binding). In variousembodiments, epitopes bound by candidate therapeutic antibodies orsiRNAs can be determined using cells isolated from non-human animals asdescribed herein.

Cells from provided non-human animals can be isolated and used on an adhoc basis, or can be maintained in culture for many generations. Invarious embodiments, cells from a provided non-human animal areimmortalized (e.g., via use of a virus) and maintained in cultureindefinitely (e.g., in serial cultures).

In some embodiments, a non-human cell is a non-human lymphocyte. In someembodiments, a non-human cell is selected from a B cell, dendritic cell,macrophage, monocyte and a T cell. In some embodiments, a non-human cellis an immature B cell, a mature naïve B cell, an activated B cell, amemory B cell, and/or a plasma cell.

In some embodiments, a non-human cell is a non-human embryonic stem (ES)cell. In some embodiments, a non-human ES cell is a rodent ES cell. Insome certain embodiments, a rodent ES cell is a mouse ES cell and isfrom a 129 strain, C57BL strain, BALB/c or a mixture thereof. In somecertain embodiments, a rodent embryonic stem cell is a mouse embryonicstem cell and is a mixture of 129 and C57BL strains. In some certainembodiments, a rodent embryonic stem cell is a mouse embryonic stem celland is a mixture of 129, C57BL and BALB/c strains.

In some embodiments, use of a non-human ES cell as described herein tomake a non-human animal is provided. In some certain embodiments, anon-human ES cell is a mouse ES cell and is used to make a mousecomprising engineered immunoglobulin κ light chain locus as describedherein. In some certain embodiments, a non-human ES cell is a rat EScell and is used to make a rat comprising engineered immunoglobulin κlight chain locus as described herein.

In some embodiments, a non-human tissue is selected from adipose,bladder, brain, breast, bone marrow, eye, heart, intestine, kidney,liver, lung, lymph node, muscle, pancreas, plasma, serum, skin, spleen,stomach, thymus, testis, ovum, and a combination thereof.

In some embodiments, an immortalized cell made, generated, produced orobtained from an isolated non-human cell or tissue as described hereinis provided.

In some embodiments, a non-human embryo made, generated, produced, orobtained from a non-human ES cell as described herein is provided. Insome certain embodiments, a non-human embryo is a rodent embryo; in someembodiments, a mouse embryo; in some embodiments, a rat embryo.

Non-human animals as described herein provide an in vivo system for thegeneration of variants of human antibody variable regions that binds apolypeptide of interest (e.g., human Vλ domain variants). Such variantsinclude human antibody variable regions having a desired functionality,specificity, low cross-reactivity to a common epitope shared by two ormore variants of a polypeptide of interest. In some embodiments,non-human animals as described herein are employed to generate panels ofhuman antibody variable regions that contain a series of variantvariable regions that are screened for a desired or improvedfunctionality.

Non-human animals as described herein provide an in vivo system forgenerating human antibody variable region libraries (e.g., a human Vλdomain library). Such libraries provide a source for heavy and/or lightchain variable region sequences that may be grafted onto different Fcregions based on a desired effector function, used as a source foraffinity maturation of the variable region sequence using techniquesknown in the art (e.g., site-directed mutagenesis, error-prone PCR,etc.) and/or used as a source of antibody components for the generationof antibody-based therapeutic molecules such as, for example, chimericantigen receptors (i.e., a molecule engineered using antibodycomponents, e.g., an scFv), multi-specific binding agents (e.g.,bi-specific binding agents) and fusion proteins (e.g., single domainantibodies, scFvs, etc.).

In some embodiments, a method of producing an antibody in a non-humananimal is provided, the method comprising the steps of (a) immunizing anon-human animal as described herein with an antigen of interest; (b)maintaining the non-human animal under conditions sufficient that thenon-human animal produces an immune response to the antigen of interest;and (c) recovering an antibody from the non-human animal, or a non-humancell, that binds the antigen of interest.

In some embodiments of a method of producing an antibody in a non-humananimal, a non-human cell is a B cell. In some embodiments of a method ofproducing an antibody in a non-human animal, a non-human cell is ahybridoma.

In some embodiments, a non-human animal is provided whose germlinegenome comprises a homozygous endogenous immunoglobulin κ light chainlocus comprising (i) human Vλ4-69, Vλ8-61, Vλ4-60, Vλ6-57, Vλ10-54,Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40,Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19,Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, Vλ3-1 orany combination thereof, (ii) human J Jλ1, Jλ2, Jλ3, Jλ6, Jλ7 or anycombination thereof, and (iii) a non-human or human Cλ gene, wherein(i)-(iii) are operably linked to each other, the non-human or human Cλgene is inserted in the place of a non-human Cκ gene of the endogenousimmunoglobulin κ light chain locus, the human Vλ gene segment(s) includehuman non-coding DNA that naturally appears adjacent to thecorresponding human Vλ gene segments in an endogenous human λ lightchain locus, and the human Jλ gene segment(s) include human non-codingDNA that naturally appears adjacent to the corresponding human Vλ genesegments in an endogenous human λ light chain locus. In some certainembodiments of a provided non-human animal, a non-human Cλ gene is orcomprises a mouse Cλ1 gene. In some certain embodiments of a providednon-human animal, a human Cλ gene is or comprises a human Cλ2 gene. Insome certain embodiments of a provided non-human animal, the endogenousimmunoglobulin κ light chain locus further comprises non-humanimmunoglobulin κ light chain enhancers Eκi and 3′ Eκ. In some certainembodiments of a provided non-human animal, the endogenousimmunoglobulin κ light chain locus includes a deletion of non-human Vκand Jκ gene segments.

In some embodiments, an antibody prepared by a method is provided,comprising the steps of: (a) providing a non-human animal as describedherein; (b) immunizing the non-human animal with an antigen of interest;(c) maintaining the non-human animal under conditions sufficient thatthe non-human animal produces an immune response to the antigen ofinterest; and (d) recovering an antibody from the non-human animal, or anon-human cell, that binds the antigen of interest, wherein the antibodyof (d) includes human V_(H) and Vλ domains.

In some embodiments of an antibody prepared by a method, a human V_(H)domain encoded by a rearranged human heavy chain variable regioncomprising a human V_(H)3-74, V_(H)3-73, V_(H)3-72, V_(H)2-70,V_(H)1-69, V_(H)3-66, V_(H)3-64, V_(H)4-61, V_(H)4-59, V_(H)1-58,V_(H)3-53, V_(H)5-51, V_(H)3-49, V_(H)3-48, V_(H)1-46, V_(H)1-45,V_(H)3-43, V_(H)4-39, V_(H)4-34, V_(H)3-33, V_(H)4-31, V_(H)3-30,V_(H)4-28, V_(H)2-26, V_(H)1-24, V_(H)3-23, V_(H)3-21, V_(H)3-20,V_(H)1-18, V_(H)3-15, V_(H)3-13, V_(H)3-11, V_(H)3-9, V_(H)1- 8,V_(H)3-7, V_(H)2-5, V_(H)7-4-1, V_(H)4-4, V_(H)1-3, V_(H)1-2 V_(H)6-1,or somatically hypermutated variant thereof.

In some embodiments of an antibody prepared by a method, a human Vλdomain encoded by a rearranged human λ light chain variable regioncomprising a human Vλ4-69, Vλ8-61, Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52,Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39,Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18,Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3 Vλ3-1, orsomatically hypermutated variant thereof.

In some embodiments, a non-human animal, non-human cell or non-humantissue as described herein is provided for use in the manufacture and/ordevelopment of a drug (e.g., an antibody or fragment thereof) fortherapy or diagnosis.

In some embodiments, a non-human animal, non-human cell or non-humantissue as described herein is provided for use in the manufacture of amedicament for the treatment, prevention or amelioration of a disease,disorder or condition.

In some embodiments, use of a non-human animal, non-human cell ornon-human tissue as described herein in the manufacture and/ordevelopment of a drug or vaccine for use in medicine, such as use as amedicament, is provided.

In some embodiments, use of a non-human animal or cell as describedherein in the manufacture and/or development of an antibody or fragmentthereof is provided.

Non-human animals as described herein provide an in vivo system for theanalysis and testing of a drug or vaccine. In various embodiments, acandidate drug or vaccine may be delivered to one or more non-humananimals as described herein, followed by monitoring of the non-humananimals to determine one or more of the immune response to the drug orvaccine, the safety profile of the drug or vaccine, or the effect on adisease or condition and/or one or more symptoms of a disease orcondition. Exemplary methods used to determine the safety profileinclude measurements of toxicity, optimal dose concentration, antibody(i.e., anti-drug) response, efficacy of the drug or vaccine and possiblerisk factors. Such drugs or vaccines may be improved and/or developed insuch non-human animals.

Vaccine efficacy may be determined in a number of ways. Briefly,non-human animals as described herein are vaccinated using methods knownin the art and then challenged with a vaccine or a vaccine isadministered to already-infected non-human animals. The response of anon-human animal(s) to a vaccine may be measured by monitoring of,and/or performing one or more assays on, the non-human animal(s) (orcells isolated therefrom) to determine the efficacy of the vaccine. Theresponse of a non-human animal(s) to the vaccine is then compared withcontrol animals, using one or more measures known in the art and/ordescribed herein.

Vaccine efficacy may further be determined by viral neutralizationassays. Briefly, non-human animals as described herein are immunized andserum is collected on various days post-immunization. Serial dilutionsof serum are pre-incubated with a virus during which time antibodies inthe serum that are specific for the virus will bind to it. Thevirus/serum mixture is then added to permissive cells to determineinfectivity by a plaque assay or microneutralization assay. Ifantibodies in the serum neutralize the virus, there are fewer plaques orlower relative luciferase units compared to a control group.

Non-human animals as described herein produce human antibody variableregions and, therefore, provide an in vivo system for the production ofhuman antibodies for use in diagnostic applications (e.g., immunology,serology, microbiology, cellular pathology, etc.). In variousembodiments, non-human animals as described herein may be used toproduce human antibody variable regions that bind relevant antigenicsites for identification of cellular changes such as, for example,expression of specific cell surface markers indicative of pathologicalchanges. Such antibodies can be conjugated to various chemical entities(e.g., a radioactive tracer) and be employed in various in vivo and/orin vitro assays as desired.

Non-human animals as described herein provide an improved in vivo systemfor development and selection of human antibodies for use in oncologyand/or infectious diseases. In various embodiments, non-human animals asdescribed herein and control non-human animals (e.g., having a geneticmodification that is different than as described herein or no geneticmodification, i.e., wild-type) may be implanted with a tumor (or tumorcells) or infected with a virus (e.g., influenza, HIV, HCV, HPV, etc.).Following implantation of infection, non-human animals may beadministered a candidate therapeutic. The tumor or virus may be allowedsufficient time to be established in one or more locations within thenon-human animals prior to administration of a candidate therapeutic.Alternatively, and/or additionally, the immune response may be monitoredin such non-human animals so as to characterize and select potentialhuman antibodies that may be developed as a therapeutic.

Pharmaceutical Compositions

In some embodiments, an antibody, a nucleic acid, or a therapeuticallyrelevant portion thereof produced by a non-human animal disclosed hereinor derived from an antibody, a nucleic acid, or a therapeuticallyrelevant portion thereof produced by a non-human animal disclosed hereincan be administered to a subject (e.g., a human subject). In someembodiments, a pharmaceutical composition includes an antibody producedby a non-human animal disclosed herein. In some embodiments, apharmaceutical composition can include a buffer, a diluent, anexcipient, or any combination thereof. In some embodiments, acomposition, if desired, can also contain one or more additionaltherapeutically active substances.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions that aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and/or perform such modification with merely ordinary, if any,experimentation.

For example, a pharmaceutical composition provided herein may beprovided in a sterile injectable form (e.g., a form that is suitable forsubcutaneous injection or intravenous infusion). For example, in someembodiments, a pharmaceutical compositions is provided in a liquiddosage form that is suitable for injection. In some embodiments, apharmaceutical composition is provided as powders (e.g., lyophilizedand/or sterilized), optionally under vacuum, which can be reconstitutedwith an aqueous diluent (e.g., water, buffer, salt solution, etc.) priorto injection. In some embodiments, a pharmaceutical compositionsisdiluted and/or reconstituted in water, sodium chloride solution, sodiumacetate solution, benzyl alcohol solution, phosphate buffered saline,etc. In some embodiments, a powder should be mixed gently with theaqueous diluent (e.g., not shaken).

Formulations of the pharmaceutical compositions described herein may beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a diluent oranother excipient and/or one or more other accessory ingredients, andthen, if necessary and/or desirable, shaping and/or packaging theproduct into a desired single- or multi-dose unit.

In some embodiments, a pharmaceutical composition including an antibodyproduced by a non-human animal disclosed herein can be included in acontainer for storage or administration, for example, an vial, a syringe(e.g., an IV syringe), or a bag (e.g., an IV bag). A pharmaceuticalcomposition in accordance with the present disclosure may be prepared,packaged, and/or sold in bulk, as a single unit dose, and/or as aplurality of single unit doses. As used herein, a “unit dose” isdiscrete amount of the pharmaceutical composition comprising apredetermined amount of the active ingredient. The amount of the activeingredient is generally equal to the dosage of the active ingredientthat would be administered to a subject and/or a convenient fraction ofsuch a dosage such as, for example, one-half or one-third of such adosage.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the disclosure will vary,depending upon the identity, size, and/or condition of the subjecttreated and further depending upon the route by which the composition isto be administered. By way of example, the composition may comprisebetween 0.1% and 100% (w/w) active ingredient.

A pharmaceutical composition may additionally comprise apharmaceutically acceptable excipient, which, as used herein, includesany and all solvents, dispersion media, diluents, or other liquidvehicles, dispersion or suspension aids, surface active agents, isotonicagents, thickening or emulsifying agents, preservatives, solid binders,lubricants and the like, as suited to the particular dosage formdesired. Remington's The Science and Practice of Pharmacy, 21st Edition,A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, Md., 2006)discloses various excipients used in formulating pharmaceuticalcompositions and known techniques for the preparation thereof. Exceptinsofar as any conventional excipient medium is incompatible with asubstance or its derivatives, such as by producing any undesirablebiological effect or otherwise interacting in a deleterious manner withany other component(s) of the pharmaceutical composition, its use iscontemplated to be within the scope of this disclosure.

In some embodiments, a pharmaceutically acceptable excipient is at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%pure. In some embodiments, an excipient is approved for use in humansand for veterinary use. In some embodiments, an excipient is approved bythe United States Food and Drug Administration. In some embodiments, anexcipient is pharmaceutical grade. In some embodiments, an excipientmeets the standards of the United States Pharmacopoeia (USP), theEuropean Pharmacopoeia (EP), the British Pharmacopoeia, and/or theInternational Pharmacopoeia.

Pharmaceutically acceptable excipients used in the manufacture ofpharmaceutical compositions include, but are not limited to, inertdiluents, dispersing and/or granulating agents, surface active agentsand/or emulsifiers, disintegrating agents, binding agents,preservatives, buffering agents, lubricating agents, and/or oils. Suchexcipients may optionally be included in pharmaceutical formulations.Excipients such as cocoa butter and suppository waxes, coloring agents,coating agents, sweetening, flavoring, and/or perfuming agents can bepresent in the composition, according to the judgment of the formulator.

In some embodiments, a provided pharmaceutical composition comprises oneor more pharmaceutically acceptable excipients (e.g., preservative,inert diluent, dispersing agent, surface active agent and/or emulsifier,buffering agent, etc.). In some embodiments, a pharmaceuticalcomposition comprises one or more preservatives. In some embodiments,pharmaceutical compositions comprise no preservative.

In some embodiments, a pharmaceutical composition is provided in a formthat can be refrigerated and/or frozen. In some embodiments, apharmaceutical composition is provided in a form that cannot berefrigerated and/or frozen. In some embodiments, reconstituted solutionsand/or liquid dosage forms may be stored for a certain period of timeafter reconstitution (e.g., 2 hours, 12 hours, 24 hours, 2 days, 5 days,7 days, 10 days, 2 weeks, a month, two months, or longer). In someembodiments, storage of antibody compositions for longer than thespecified time results in antibody degradation.

Liquid dosage forms and/or reconstituted solutions may compriseparticulate matter and/or discoloration prior to administration. In someembodiments, a solution should not be used if discolored or cloudyand/or if particulate matter remains after filtration.

General considerations in the formulation and/or manufacture ofpharmaceutical agents may be found, for example, in Remington: TheScience and Practice of Pharmacy 21st ed., Lippincott Williams &Wilkins, 2005.

Kits

The present disclosure further provides a pack or kit comprising one ormore containers filled with at least one non-human animal, non-humancell, DNA fragment, targeting vector, or any combination thereof, asdescribed herein. Kits may be used in any applicable method (e.g., aresearch method). Optionally associated with such container(s) can be anotice in the form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals or biological products,which notice reflects (a) approval by the agency of manufacture, use orsale for human administration, (b) directions for use, and/or (c) acontract that governs the transfer of materials and/or biologicalproducts (e.g., a non-human animal or non-human cell as describedherein) between two or more entities and combinations thereof.

In some embodiments, a kit comprising a non-human animal, non-humancell, non-human tissue, immortalized cell, non-human ES cell, ornon-human embryo as described herein is provided. In some embodiments, akit comprising an amino acid (e.g., an antibody or fragment thereof)from a non-human animal, non-human cell, non-human tissue, immortalizedcell, non-human ES cell, or non-human embryo as described herein isprovided. In some embodiments, a kit comprising a nucleic acid (e.g., anucleic acid encoding an antibody or fragment thereof) from a non-humananimal, non-human cell, non-human tissue, immortalized cell, non-humanES cell, or non-human embryo as described herein is provided. In someembodiments, a kit comprising a sequence (amino acid and/or nucleic acidsequence) identified from a non-human animal, non-human cell, non-humantissue, immortalized cell, non-human ES cell, or non-human embryo asdescribed herein is provided.

In some embodiments, a kit as described herein for use in themanufacture and/or development of a drug (e.g., an antibody or fragmentthereof) for therapy or diagnosis is provided.

In some embodiments, a kit as described herein for use in themanufacture and/or development of a drug (e.g., an antibody or fragmentthereof) for the treatment, prevention or amelioration of a disease,disorder or condition is provided.

Other features of certain embodiments will become apparent in the courseof the following descriptions of exemplary embodiments, which are givenfor illustration and are not intended to be limiting thereof.

EXAMPLES

The following examples are provided so as to describe to the skilledartisan how to make and use methods and compositions described herein,and are not intended to limit the scope of what the inventors of thepresent disclosure regard as their invention. Unless indicatedotherwise, temperature is indicated in Celsius and pressure is at ornear atmospheric.

Example 1. Construction of a Targeting Vectors for Generating a RodentExpressing at Least One Lambda Light Chain from a Kappa Light ChainLocus Example 1.1. Engineering a Targeting Vector Comprising a RodentLambda Constant Region

This example illustrates exemplary methods of constructing a targetingvector for insertion into the genome of a non-human animal such as arodent (e.g., a mouse). Furthermore, this example demonstratesproduction of a non-human animal whose germline genome comprises anengineered immunoglobulin κ light chain locus. In particular, thisexample demonstrates construction of a targeting vector for engineeringan endogenous immunoglobulin κ light chain locus in a rodent so that therodent expresses and/or produces antibodies that include immunoglobulinλ light chains having human variable regions and non-humanimmunoglobulin λ constant (Cλ) regions from said immunoglobulin κ lightchain locus in the germline genome of the non-human animal. As describedbelow in Example 2, DNA fragments containing multiple human Jλ (e.g.,Jλ1, Jλ2, Jλ3, Jλ6 and Jλ7) coding sequences and a rodent Cλ (e.g., amouse Cλ1) coding sequence are inserted into an endogenous rodentimmunoglobulin κ light chain locus. In particular exemplifiedembodiments, a mouse Cλ1 gene is inserted in the place of a mouse Cκgene and in operable linkage with rodent Igκ enhancers (e.g., Eκi and3′Eκ). An exemplary strategy for creation of a targeting vector forgenerating an engineered immunoglobulin κ light chain locus in a rodentcharacterized by the presence of a plurality of human Vλ and Jλ genesegments operably linked to a rodent (e.g., mouse) Cλ gene and operablylinked to endogenous Igκ enhancers is set forth in FIG. 1 (FIG. 1A:initial steps of construction of targeting vector; FIG. 1B: additionalsubsequent steps of construction of a targeting vector; a humanimmunoglobulin κ light chain sequence between human Vλ and Jλ genesegments is indicated by an open bar filed with wide downward diagonallines (e.g., see U.S. Pat. Nos. 9,006,511, 9,035,128, 9,066,502,9,150,662 and 9,163,092), lox: lox2372; NEO: Neomycin resistance gene(neo^(R)) under transcriptional control of a ubiquitin promoter, HYG:Hygromycin resistance gene (hyg^(R)) under transcriptional control of aubiquitin promoter, Spec: Spectinomycin resistance gene (Spec^(R)), R6κ:R6K origin of replication).

A targeting vector containing human Jλ and rodent Cλ coding sequencesfor insertion into a rodent immunoglobulin κ light chain locus wascreated using VELOCIGENE® technology (see, e.g., U.S. Pat. No. 6,586,251and Valenzuela et al., 2003, Nature Biotech. 21(6):652-9; incorporatedherein by reference in their entireties) and molecular biologytechniques known in the art. Those of ordinary skill, reading thepresent example, will appreciate that the described technologies andapproach can be employed to utilize any human Jλ and any Cλ codingsequences, or combination of coding sequences (or sequence fragments) asdesired.

Briefly, a 2.7 kb DNA fragment containing human Jλ gene segments Jλ1,Jλ2, Jλ3, Jλ6 and Jλ7 and unique 5′ and 3′ overlap regions correspondingto a human Vκ-Jκ genomic (non-coding) sequence and a genomic sequence 5′of a mouse Cκ gene, respectively, was made by de novo DNA synthesis (pA;FIG. 1A, top left, Blue Heron Biotech, Bothell, Wash.). Variousrestriction enzyme recognition sites were included in the DNA fragmentto facilitate subsequent cloning of selection markers and other DNAfragments (described below). The DNA fragment was uniquely designed tocontain non-coding human Jκ sequences juxtaposed with human Jλ codingsequences and human Jλ recombination signal sequences (RSS). As in knownin the art, an RSS consists of a conserved block of seven nucleotides(heptamer) followed by a spacer either 12 or 23 base pairs in length andfollowed by a second conserved block of nine nucleotides (nonamer).Thus, an RSS has a configuration of 7-12-9 (12RSS) or 7-23-9 (23RSS)depending on the associated gene segment (see, e.g., FIG. 5.4 in Murphy,Kenneth, et al. “Chapter 5.” Janeway's Immunobiology, 8th ed., GarlandScience/Taylor & Francis Group, L L C, 2012, which is incorporatedherein by reference in its entirety). In particular, human Jλ genesegments (i.e., human Jλ coding sequences) and their associated 12RSSwere substituted in the place of human Jκ gene segments (i.e., human Jκcoding sequences) and their associated 23RSS. Thus, this fragmentcontained human Jλ and Jκ DNA sequences. Inclusion of such sequences intargeting vectors described herein can provide for (or promote)efficient joining of human Vλ and Jλ gene segments within an engineeredrodent Igκ light chain locus.

Plasmid A (pA) was digested with AgeI and EcoRI and ligated to aNeomycin selection cassette (i.e., a Neo^(R) gene under control of aubiquitin promoter flanked by lox2372 sites) containing compatible endsto create plasmid B (pB) (FIG. 1A). Separately, unique DNA fragmentscontaining a mouse Igκ intronic enhancer (Ei), a mouse Cλ1 gene (fromBAC clone RP23-60e14), a DNA fragment containing 316 bp of sequenceimmediately downstream of a mouse Cκ coding sequence and 80 bp ofoverlap sequence to facilitate isothermal assembly, and restrictionenzyme recognition sites (NotI, MluI) to facilitate subsequent cloningsteps, and an R6K-Spec (a Spectinomycin Adenylytransferase gene and aR6K origin of replication) were amplified by polymerase chain reaction(PCR) and combined together by isothermal assembly (see, e.g., Gibson,D. G. et. al., 2009, Nat. Meth. 6(5):343-5; Gibson, D. G. et al., 2010,Nat. Meth. 7:901-903; incorporated herein by reference in theirentireties) to create plasmid C (pC), which was subsequently digestedwith NotI and MluI and ligated to a Hygromycin selection cassette (i.e.,a Hyg^(R) gene under control of a ubiquitin promoter flanked by loxPsites) containing compatible ends to create plasmid D (pD; FIG. 1A, topand middle right). This resulting plasmid (pD; FIG. 1A, middle) was thendigested with PI-SceI and AscI and ligated with plasmid B (pB)containing compatible ends (FIG. 1A, bottom) to generate plasmid E (pE).

In a next step, a targeting vector containing human Vλ and Jλ genesegments operably linked to a mouse Cκ gene and a human immunoglobulin κlight chain sequence positioned between the human Vλ and Jλ genesegments (see, e.g., U.S. Pat. No. 9,006,511, which is incorporatedherein by reference in its entirety) was separately digested with NotIand religated to remove the human Vλ region including a humanimmunoglobulin κ sequence (FIG. 1B, top), which resulted in a deletionof ˜137 kb. The resulting construct (construct F) was combined withplasmid E (pE) using a CRISPR/Cas9 isothermal assembly method (see,e.g., U.S. Pat. No. 9,738,897, and U.S. Publication No. 2016/0145646;incorporated herein by reference in their entireties) so that the humanJκ region with human Jλ-12RSS coding sequence (CDS) was operably linkedwith a human Vκ-Jκ intergenic (non-coding) region (see, e.g., U.S. Pat.No. 9,006,511, which is incorporated herein by reference in itsentirety) and a mouse Igκ 3′ enhancer (FIG. 1B). Positive bacterialclones were selected on media containing Kanamycin, Hygromycin andSpectinomycin. The resulting targeting vector (construct G) contained,from 5′ to 3′, a loxP recognition site, a NotI site, a human Vκ-Jκintergenic sequence (see, e.g., U.S. Pat. No. 9,006,511, which isincorporated by reference herein in its entirety), a Neomycin selectioncassette flanked by lox2372 recognition sites, a human Jκ region withfive human Jλ gene segments and their respective 12RSS, a mouseimmunoglobulin κ intronic enhancer (Eiκ), a mouse Cλ1 gene, a Hygromycinselection cassette flanked by loxP recognition sites, a mouseimmunoglobulin κ 3′ enhancer (3′ Eκ) and a Spectinomycin selectioncassette (FIG. 1B).

Example 1.2. Engineering a Targeting Vector Comprising a Human LambdaConstant Region

This example illustrates exemplary methods of constructing a targetingvector for insertion into the genome of a non-human animal such as arodent (e.g., a mouse). Furthermore, this example demonstratesproduction of a non-human animal whose germline genome comprises anengineered immunoglobulin κ light chain locus. In particular, thisexample demonstrates construction of a targeting vector for engineeringan endogenous immunoglobulin κ light chain locus in a rodent so that therodent expresses and/or produces antibodies that include immunoglobulinλ light chains having human variable regions and human immunoglobulin λconstant (Cλ) regions from said immunoglobulin κ light chain locus inthe germline genome of the non-human animal. As described below inExample 2, DNA fragments containing multiple human Jλ (e.g., Jλ1, Jλ2,Jλ3, Jλ6 and Jλ7) coding sequences and a human Cλ (e.g., a human Cλ2)coding sequence are inserted into an endogenous rodent immunoglobulin κlight chain locus. In particular, a human Cλ2 gene is inserted in theplace of a mouse Cκ gene and in operable linkage with rodentimmunoglobulin κ enhancers (e.g., Eiκ and 3′Eκ). An exemplary strategyfor creation of a targeting vector is set forth in FIG. 3.

A targeting vector containing human Jλ and human Cλ coding sequences forinsertion into a rodent Igκ light chain locus was created usingVELOCIGENE® technology (see, e.g., U.S. Pat. No. 6,586,251 andValenzuela et al., 2003, Nature Biotech. 21(6):652-9; incorporatedherein by reference in their entireties) and molecular biologytechniques known in the art. Those of ordinary skill, reading thepresent example, will appreciate that the described approach andtechnologies can be employed to utilize any human Jλ and Cλ codingsequences, or combination of coding sequences (or sequence fragments) asdesired.

Briefly, an 871 bp DNA fragment containing a human Cλ coding sequenceand unique 5′ and 3′ overlap regions corresponding to genomic sequences5′ and 3′ of a mouse Cκ gene, respectively, was made by de novo DNAsynthesis (pH; FIG. 3, top left, Blue Heron Biotech, Bothell, Wash.).Various restriction enzyme recognition sites were included in the DNAfragment to allow for subsequent cloning of selection markers and otherDNA fragments (described below). Plasmid H (pH) was digested with AgeIand XhoI and ligated to a Hygromycin selection cassette (i.e., a Hyg^(R)gene under control of a ubiquitin promoter flanked by loxP sites)containing compatible ends to create plasmid J (pJ; FIG. 3). Anintermediate construct (construct K, generated from construct F andplasmid B using Cas9 and isothermal assembly) containing an engineeredhuman Jκ region with human Jλ coding sequences (described above)operably linked to a mouse Cκ gene and mouse Igκ enhancers was combinedwith plasmid J using a CRISPR/Cas9 isothermal assembly method (see,e.g., U.S. Pat. No. 9,738,897, and U.S. Publication No. 2016/0145646;incorporated herein by reference in their entireties) so that the humanJκ region with human Jλ-12RSS coding sequence (CDS) was operably linkedwith the human Cλ2 coding sequence of plasmid J (FIG. 3). Positivebacterial clones were selected on media containing Kanamycin, Hygromycinand Spectinomycin. The resulting targeting vector (construct L)contained, from 5′ to 3′, a loxP recognition site, a NotI site, a humanVκ-Jκ intergenic sequence (see, e.g., U.S. Pat. No. 9,006,511, which isincorporated by reference herein in its entirety), a Neomycin selectioncassette flanked by lox2372 recognition sites, a human Jκ region withfive human Jλ gene segments and their respective 12RSS, a mouse Igκintronic enhancer (Eiκ), a human Cλ2 gene, a Hygromycin selectioncassette flanked by loxP recognition sites, a mouse immunoglobulin κ 3′enhancer (3′ Eκ) and a Spectinomycin selection cassette (FIG. 3).

Example 2. Generation of Rodents Having an Engineered Light Chain Locus

This example demonstrates production of non-human animals (e.g.,rodents) whose germline genome comprises an endogenous immunoglobulin κlight chain locus comprising insertion of a plurality of human Vλ and Jλgene segments and a rodent or human Cλ gene, which human Vλ and Jλ genesegments are operably linked to said rodent or human Cλ gene, and whichrodent or human Cλ gene is inserted in the place of a rodent Cκ gene ofan endogenous immunoglobulin κ light chain locus. Such non-human animalsare characterized, in some embodiments, by expression of immunoglobulinλ light chains (variable and constant domains) from an endogenousimmunoglobulin κ light chain locus.

Targeted insertion of targeting vectors described in Examples 1.1 and1.2 were confirmed by polymerase chain reaction. Targeted BAC DNA,confirmed by polymerase chain reaction, was introduced into F¹ hybrid(C57BL6NTac/129S6SvEvTac) mouse embryonic stem (ES) cells viaelectroporation followed by culturing in selection medium.

ES cells used for electroporation of construct G (mouse Cλ1) had agermline genome that included a heterozygous immunoglobulin κ lightchain locus containing a plurality of human Vλ and Jλ gene segmentsoperably linked to a rodent immunoglobulin κ light chain constant regiongene including rodent immunoglobulin κ light chain enhancers, and ahuman immunoglobulin κ light chain sequence positioned between the humanVλ and Jλ gene segments and one wild-type rodent immunoglobulin κ lightchain locus. ES cells before and after electroporation are depicted inFIG. 2A (1741HET: a rodent ES cell clone having a genome heterozygousfor an engineered immunoglobulin κ light chain locus containing human Vλand Jλ gene segments operably linked to a rodent immunoglobulin κ lightchain constant region gene including rodent immunoglobulin κ light chainenhancers, and a human immunoglobulin κ light chain sequence positionedbetween the human Vλ and Jλ gene segments indicated by an open bar filedwith wide downward diagonal lines, and wild-type immunoglobulin heavyand λ light chain loci, e.g., see U.S. Pat. Nos. 9,006,511, 9,035,128,9,066,502, 9,150,662 and 9,163,092, which are hereby incorporated byreference in their entireties; 6557HET: a mouse ES cell clone afterinsertion of construct G resulting in a genome heterozygous for anengineered immunoglobulin κ light chain locus including rodentimmunoglobulin κ light chain enhancers, which engineered immunoglobulinκ light chain locus is characterized by the presence of a plurality ofhuman Vλ and Jλ gene segments, which human Jλ gene segments arecontained within a human Jκ region sequence with human Jλ gene segmentcoding sequences and human Jλ 12RSS in place of the corresponding humanJκ gene segment coding sequences and human Jκ 23RSS, and which human Vλand Jλ gene segments are operably linked to a rodent immunoglobulin λlight chain constant region gene (e.g., mCλ1); lox: lox2372; NEO:Neomycin resistance gene (neo^(R)) under transcriptional control of aubiquitin promoter; HYG: Hygromycin resistance gene (hyg^(R)) undertranscriptional control of a ubiquitin promoter; locations of selectedprimer/probe sets for screening ES cells clones are indicated near thelocations of regions within the engineered Igκ light chain locusdetected in an assay described below).

ES cells used for electroporation of construct L (human Cλ2) had agermline genome that included a heterozygous immunoglobulin κ lightchain locus containing a plurality of human Vλ and Jλ gene segmentsoperably linked to a rodent immunoglobulin κ light chain constant regiongene including rodent immunoglobulin κ light chain enhancers, and ahuman immunoglobulin κ light chain sequence positioned between the humanVλ and Jλ gene segments and one wild-type rodent Igκ locus. ES cellsbefore and after electroporation are depicted in FIG. 4A (1741HET:supra; 20029HET: a mouse ES cell clone after insertion of a targetingvector having a genome heterozygous for an engineered Igκ light chainlocus including rodent immunoglobulin κ light chain enhancers, whichengineered Igκ light chain locus is characterized by the presence of aplurality of human Vλ and Jλ gene segments, which human Jλ gene segmentsare contained within a human Jκ region sequence with human Jλ genesegment coding sequences and human Jλ 12RSS in place of thecorresponding human Jκ gene segment coding sequences and human Jκ 23RSS,and which human Vλ and Jλ gene segments are operably linked to a humanIgλ light chain constant region gene (e.g., hCλ2); lox: lox2372; NEO:Neomycin resistance gene (neo^(R)) under transcriptional control of aubiquitin promoter; HYG: Hygromycin resistance gene (hyg^(R)) undertranscriptional control of a ubiquitin promoter; locations of selectedprimer/probe sets for screening ES cells clones are indicated near thelocations of regions within the engineered Igκ light chain locusdetected in an assay described below).

Drug-resistant colonies were picked 10 days after electroporation andscreened by TAQMAN™ and karyotyping for correct targeting as previouslydescribed (Valenzuela et al., supra; Frendewey, D. et al., 2010, MethodsEnzymol. 476:295-307; incorporated herein by reference in theirentireties). Table 1 sets forth exemplary primers/probes sets used forscreening positive ES cell clones (F: forward; R: reverse; P: probe;GOA: gain of allele; LOA: loss of allele; WT: wild-type).

The VELOCIMOUSE® method (DeChiara, T. M. et al., 2010, Methods Enzymol.476:285-294; DeChiara, T. M., 2009, Methods Mol. Biol. 530:311-324;Poueymirou et al., 2007, Nat. Biotechnol. 25:91-99; incorporated hereinby reference in their entireties), in which targeted ES cells wereinjected into uncompacted 8-cell stage Swiss Webster embryos, was usedto produce healthy fully ES cell-derived F0 generation mice heterozygousfor the engineered Igκ light chain locus (FIGS. 2A and 4A). F0generation heterozygous mice were crossed with C57Bl6/NTac mice togenerate F1 heterozygotes that were intercrossed to produce homozygousF2 generation animals for phenotypic analyses.

Alternatively, murine ES cells bearing an engineered immunoglobulin κlocus as described above can be modified to remove one or more selectioncassettes introduced with a targeting vector as desired (FIG. 2B:6557HET: supra; 6558HET: a mouse ES cell clone afterrecombinase-mediated excision of Neomycin and Hygromycin selectioncassettes inserted after homologous recombination with a targetingvector; FIG. 4B: 20029HET: supra; 20030HET: a mouse ES cell clone afterrecombinase-mediated excision of Neomycin and Hygromycin selectioncassettes inserted after homologous recombination with a targetingvector. Cre: Cre recombinase). For example, the Neomycin and Hygromycincassette introduced by the targeting vectors may be removed inengineered ES cells (or embryos) by transient recombinase expression orby breeding to a recombinase-expressing genetically engineered strain(see e.g., Lakso, M. et al., 1992, Proc. Natl. Acad. Sci. USA 89:6232-6; Orban, P. C. et al., 1992, Proc. Natl. Acad. Sci. U.S.A.89:6861-5; Gu, H. et al., 1993, Cell 73(6):1155-64; Araki, K. et al.,1995, Proc. Natl. Acad. Sci. U.S.A. 92:160-4; Dymecki, S. M., 1996,Proc. Natl. Acad. Sci. U.S.A. 93(12):6191-6; all of which areincorporated herein by reference in their entireties).

Taken together, this example illustrates the generation of a rodent(e.g., a mouse) whose germline genome comprises an engineered Igκ lightchain locus characterized by the presence of a plurality of human Vλ andJλ gene segments operably linked to a rodent or human Cλ gene, whichrodent or human Cλ gene is inserted in the place of a rodent Cκ gene ofan endogenous Igκ light chain locus. An engineered Igκ light chain locusas described includes plurality of human Vλ and Jλ gene segments in anon-endogenous arrangement. The strategy described herein for insertinghuman Vλ and Jλ gene segments, and a rodent or human Cλ gene into theplace of a rodent Cκ gene, enables the construction of a rodent thatexpresses antibodies that exclusively contain human Vλ domains. It wasunclear if such an engineered Igκ light chain locus that includesexclusively λ gene segments (outside of the endogenous λ locus, in anon-endogenous orientation) would be able to produce functional lightchains. As described herein, such human Vλ domains are expressed fromendogenous Igκ light chain loci in the germline genome of providedrodents.

TABLE 1Representative primer/probe sets for screening positive ES cell clonesName Sequence (5′-3′) Assay mIgKC-1 FGTGGAAGATTGATGGCAGTGAAC (SEQ ID NO: 25) LOA RGTGCTGCTCATGCTGTAGGT (SEQ ID NO: 26) PAAATGGCGTCCTGAACAGTTGGACTGA (SEQ ID NO: 27) mIgKC-2 FCCATCCAGTGAGCAGTTAACATC (SEQ ID NO: 28) LOA RTGTCGTTCACTGCCATCAATC (SEQ ID NO: 29) PAGGTGCCTCAGTCGTGTGCTTC (SEQ ID NO: 30) mIgKLC1-1 FGGAGCCCTTCCTTGTTACTTCA (SEQ ID NO: 31) GOA-6557 RAGGTGGAAACAGGGTGACTGATG (SEQ ID NO: 32) PTCCTCTGTGCTTCCTTCCTCAGGC (SEQ ID NO: 33) mIgKLC1-2 FTCCTTGTTACTTCATACCATCCTCT (SEQ ID NO: 34) GOA-6557 RAGGGTGACTGATGGCGAAGACT (SEQ ID NO: 35) PTTCCTTCCTCAGGCCAGCCC (SEQ ID NO: 36) hIgKLJ-1 FGAGGCTTGCTGAGCTTTCAG (SEQ ID NO: 37) GOA RAGGACGGTCAGCTTGGTC (SEQ ID NO: 38) PTATGAGCCTGTGTCACAGTGTTGGG (SEQ ID NO: 39) hIgKLJ-2 FGCTGACCCAGGACTCTGTTC (SEQ ID NO: 40) GOA RTCCCAGTTCCGAAGACATAACAC (SEQ ID NO: 41) PCCCTTTGGTGAGAAGGGTTTTGGTC (SEQ ID NO: 42) hIgLC2-1 FTACGCGGCCAGCAGCTAT (SEQ ID NO: 43) GOA-20029 RTGGCAGCTGTAGCTTCTGT (SEQ ID NO: 44) PCTGAGCCTGACGCCTGAGCAG (SEQ ID NO: 45) 1561hJ1 FTCAACCTTTCCCAGCCTGTCT (SEQ ID NO: 46) LOA RCCCCAGAGAGAGAAAACAGATTTT (SEQ ID NO: 47) PACCCTCTGCTGTCCCT (SEQ ID NO: 49) Neo FGGTGGAGAGGCTATTCGGC (SEQ ID NO: 50) GOA RGAACACGGCGGCATCAG (SEQ ID NO: 51) PTGGGCACAACAGACAATCGGCTG (SEQ ID NO: 52) Hyg FTGCGGCCGATCTTAGCC (SEQ ID NO: 53) GOA RTTGACCGATTCCTTGCGG (SEQ ID NO: 54) PACGAGCGGGTTCGGCCCATTC (SEQ ID NO: 55) 1468h2 FGGGCTACTTGAGGACCTTGCT (SEQ ID NO: 56) Parental RGACAGCCCTTACAGAGTTTGGAA (SEQ ID NO: 57) PCAGGGCCTCCATCCCAGGCA (SEQ ID NO: 58) 1525hk-VJ1 FATCTCCCTACTTCCTGGCTAATG (SEQ ID NO: 59) Parental RGCTTGGAACCTGATTGGTTGTC (SEQ ID NO: 60) PAGCCTTGATCCTTGGGAATCCAGGACA (SEQ ID NO: 61) mIgKd2 FGCAAACAAAAACCACTGGCC (SEQ ID NO: 62) WT RGGCCACATTCCATGGGTTC (SEQ ID NO: 63) PCTGTTCCTCTAAAACTGGACTCCACAGTAAATGGAAA (SEQ ID NO: 64)

Example 3. Characterization of Rodents Having an EngineeredImmunoglobulin Light Chain Locus Example 3.1. Phenotypic Assessment ofImmune Cells in Rodents Having an Engineered Immunoglobulin Light ChainLocus

This example demonstrates the characterization of various immune cellpopulations in rodents (e.g., mice) engineered to contain a plurality ofhuman Vλ and Jλ gene segments operably linked to a rodent Cλ gene, androdent immunoglobulin κ light chain enhancer and regulatory regions,within an endogenous immunoglobulin κ light chain locus. In particular,this example specifically demonstrates that rodents having engineeredimmunoglobulin κ light chain loci described herein display a uniquelight chain expression profile as compared to wild-type rodents. Thisexample also demonstrates that provided rodents express a broadrepertoire of human V, regions from the engineered immunoglobulin κlight chain locus.

Briefly, spleens and femurs were harvested from wild-type (WT, 75%C57BL/6NTac 25% 129SvEvTac) and 6558HO (homozygous LiK, 75% C57BL/6NTac25% 129SvEvTac) mice. Bone marrow was collected from femurs by flushingwith 1× phosphate buffered saline (PBS, Gibco) with 2.5% fetal bovineserum (FBS). Red blood cells from spleen and bone marrow preparationswere lysed with ACK lysis buffer (Gibco) followed by washing with 1×PBSwith 2.5% FBS.

Isolated cells (1×10⁶) were incubated with selected antibody cocktailsfor 30 min at +4° C.: anti-mIgK-FITC (187.1, BD Biosciences),anti-mIgλ-PE (RML-42, BioLegend; 1060-09, Southern Biotech),anti-mIgλ-FITC (106002, Bio-Rad; ABIN303989, Antibodies-online),anti-mouse IgM-PeCy7 (II/41, eBioscience), anti-mouse IgD-PerCP/Cy5.5(11-26c.2a, BioLegend), anti-mouse CD3-Pacific Blue (17A2, BioLegend),anti-mouse B220-APC (RA3-6B2, eBioscience), anti-mouse CD19-APC-H7 (ID3,BD Biosciences). Following staining, cells were washed and fixed in 2%formaldehyde. Data acquisition was performed on a BD LSRFORTESSA™ flowcytometer and analyzed with FLOWJO™ software. Representative results areset forth in FIGS. 5-7.

As shown in FIGS. 5 and 6, LiK mice demonstrate similar distributions ofCD19⁺ and immature/mature B cells as compared to wild-type mice in thespleen and bone marrow compartments, respectively. However, LiK micedemonstrate a unique light chain expression as compared to wild-typemice in that only Igλ⁺ expression was observed in these mice (FIG. 7).In particular, >90% of CD19⁺ B cells in LiK mice express immunoglobulin,light chain thereby confirming proper recombination and expression atthe engineered immunoglobulin κ locus. As expected given these mice lacka mouse Cκ gene, LiK mice demonstrate no detectable immunoglobulin κexpression by flow cytometry (i.e., the anti-mIgκ antibody detects theconstant region). Similar levels of immunoglobulin λ light chainexpression were observed from additional LiK mice littermates (data notshown). Expression of human Vλ regions in the context of a mouse Cλregion from the LiK locus was confirmed by, among other things,immunoglobulin repertoire analysis using Next Generation Sequencingtechniques (described in Example 3.2 below).

Example 3.2. Immunoglobulin Repertoire in Rodents Having an EngineeredImmunoglobulin Light Chain Locus

Usage of human antibody genes (i.e., VDJ gene segments) in theengineered rodent strain described above was determined by NextGeneration Sequencing antibody repertoire analysis. In particular,RT-PCR sequencing was conducted on RNA isolated from splenocytes of micehomozygous for the LiK locus (6558 HO) to confirm correct transcriptionand recombination of the LiK locus. A representative illustration of arearranged LiK locus is set forth in FIG. 12 (LiK locus: engineeredimmunoglobulin κ light chain locus as described herein; rearranged LiKlocus: representative rearrangement of engineered immunoglobulin κ lightchain locus (referred to herein as “LiK locus”) resulting in human Vλ-Jλrecombination; rearranged LiK mRNA: representative transcription andmRNA processing of rearranged LiK locus).

Briefly, splenic B cells were positively enriched from total splenocytesby magnetic cell sorting using mouse anti-CD19 magnetic beads and MACS®columns (Miltenyi Biotech). Total RNA was isolated from purified splenicB cells using an RNeasy Plus RNA isolation kit (Qiagen) according tomanufacturer's specifications. Reverse transcription was performed togenerate cDNA containing immunoglobulin, constant region gene sequence,using a SMARTer™ RACE cDNA Amplification Kit (Clontech) andimmunoglobulin, specific primers (see below). During this process, a DNAsequence, reverse compliment to 3′ of a template switching (TS) primer,was attached to the 3′ end of newly synthesized cDNAs. PurifiedIgλ-specific cDNAs were then amplified by a 1^(st) round PCR reactionusing the TS specific primer and reverse primers specific to sequencesof mouse Cλ1. PCR products ranging from ˜450-700 bp were isolated usingPippin Prep (SAGE Science) and then these fragments were furtheramplified by a 2^(nd) round PCR reaction. Table 2 sets forth thesequences of selected primers used for repertoire library construction(for: forward primer; rev: reverse primer). PCR products ranging from˜400 bp-700 bp were isolated, purified, and quantified by qPCR using aKAPA Library Quantification Kit (KAPA Biosystems) before loading onto aMiseq sequencer (Illumina) for sequencing using Miseq Reagent Kits v3(2×300 cycles).

For bioinformatic analysis, Raw Illumina sequences were de-muliplexedand filtered based on quality, length and match to correspondingconstant region gene primer. Overlapping paired-end reads were mergedand analyzed using custom in-house pipeline. The pipeline used localinstallation of IgBLAST (NCBI, v2.2.25⁺) to align rearranged light chainsequences to human germline Vλ and Jλ gene segment database, and denotedproductive and non-productive joins along with the presence of stopcodons. CDR3 sequences and expected non-template nucleotides wereextracted using boundaries as defined in International ImmunogeneticsInformation System (IMGT).

TABLE 2 Representative primers for repertoire library constructionPrimer Name Sequence (5′-3′) TS primerCACCATCGAT GTCGACACGC CTAGGG (SEQ ID NO: 65) IgλCCACCAGTGTG GCCTTGTTAG TCTC (SEQ ID NO: 66) (RT primer) IgλCACACTCTTTC CCTACACGAC GCTCTTCCGA TCTCAGGGTG ACTGATGGCG (1^(st) PCR)AAGAC (SEQ ID NO: 67) TS specificGTGACTGGAG TTCAGACGTG TGCTCTTCCG ATCTCACCAT CGATGTCGAC (1^(st) PCR)ACGCCTA (SEQ ID NO: 68) for (2^(nd) PCR)AATGATACGG CGACCACCGA GATCTACAC XXXXXX ACACTCTTTCCCTACACGAC GCTCTTCCGA TCT (SEQ ID NO: 69) rev (2^(nd) PCR)CAAGCAGAAG ACGGCATACG AGAT XXXXXX GTGACTGGAG TTCAGACGTGTGCTCTTCCG ATCT (SEQ ID NO: 70)

The majority of the functional human Vλ and Jλ gene segments included inthe LiK locus in engineered mice exemplified herein were represented inthe expressed antibody repertoire of LiK mice comprising a plurality ofhuman Vλ and Jλ gene segments operably linked to a rodent Cλ gene at theendogenous kappa locus (data not shown). Overall, the inventors observedthat the B cells of LiK mice expressed antibodies having light chainsexpressed from the LiK locus as expected. No altered splicing products,insertions, deletions or otherwise unexpected mutations were observed inthe transcripts analyzed. These results confirm that recombination atthe LiK locus generates functional light chains as part of the antibodyrepertoire of these mice. Similar analysis was performed in micecomprising a plurality of human Vλ and Jλ gene segments operably linkedto a human Cλ gene at the endogenous kappa locus, where the expressionof a plurality of human Vλ and Jλ gene segments was detected (data notshown).

Example 3.3. Antibody Expression in Rodents Having an Engineered LightChain Locus

This example demonstrates expression of antibodies (e.g., IgG) fromnon-human animals, which antibodies contain light chains characterizedby the presence of human Vλ regions and rodent or human Cλ regions, andwhich light chains are expressed from an engineered endogenous rodentimmunoglobulin κ light chain locus. Among other things, this examplespecifically demonstrates expression of IgG antibodies (in dimeric andmonomeric forms) in the serum of non-human animals (e.g., rodents) whosegermline genome comprises an endogenous immunoglobulin κ light chainlocus comprising insertion of one or more human Vλ gene segments, one ormore human Jλ gene segments and a rodent Cλ gene, which human Vλ and Jλgene segments are operably linked to said rodent Cλ gene, and whichrodent Cλ gene is inserted in the place of a rodent Cκ gene of anendogenous rodent Igκ light chain locus.

Blood was drawn from wild-type (WT, 75% C57BL/6NTac 25% 129SvEvTac) and6558 homozygous (“LiK”, 75% C57BL/6NTac 25% 129SvEvTac) mice. Serum wasseparated from blood using Eppendorf tubes centrifuged at 9000 rpm forfive minutes at 4° C. Collected serum was used for immunoblotting toidentify expression of IgG antibodies.

Mouse sera were diluted 1:100 or 1:500 in PBS (without Cλ2⁺ and Mg2⁺)and run on 4-20% Novex Tris-Glycine gels under reducing and non-reducingconditions. Gels were transferred to Polyvinylidene difluoride (PVDF)membranes according to manufacturer's specifications. Blots were blockedovernight with 5% nonfat milk in Tris-Buffered Saline with 0.05%Tween-20 (TBST, Sigma). PVDF membranes were exposed to primary antibody(goat anti-mIgG1 conjugated to HRP, Southern Biotech) diluted 1:1000 in0.1% nonfat milk in TBST for one hour at room temperature. Blots werewashed four times for ten minutes per wash and developed for fiveminutes with Amersham ECL Western Blotting Detection Reagent (GEHealthcare Life Sciences) according to manufacturer's specifications.Blots were then imaged using GE Healthcare ImageQuant LAS-4000 CooledCCD Camera Gel Documentation System. Images were captured at 15 secondintervals until 20 images were captured or images were fully exposed,whichever came first. Representative results are set forth in FIG. 13(lane numbers are indicated at the top of each gel image and laneassignments are the same for both images; top left: reduced samples;bottom left: non-reduced samples; LiK HO: 6558 homozygous; WT:wild-type; molecular weights are indicated on the left of each gelimage).

As shown in FIG. 13, the size of IgG antibodies expressed in LiK mice issimilar to the size observed for IgG antibodies expressed in wild-typemice, which demonstrates that LiK mice produce functional antibodiesthat bind antigen and can be used as an in vivo system for theproduction of human antibodies and human antibody components for use inthe treatment of human disease(s).

Example 4. Generation and Characterization of Rodents Comprising SeveralEngineered Immunoglobulin Loci

LiK rodents as described herein are separately bred with multipleengineered rodent strains over multiple breedings using techniques knownin the art to establish rodents strains containing the followingengineered immunoglobulin loci: (1) a rodent strain homozygous forhumanized immunoglobulin heavy chain (see, e.g., U.S. Pat. Nos.8,642,835 and 8,697,940, each of which is incorporated herein byreference in its entirety), homozygous for an immunoglobulin κ lightchain locus comprising human Vλ and Jλ gene segments operably linked toa Cλ gene as described herein and homozygous for an inactivatedendogenous immunoglobulin λ light chain locus (see, e.g., U.S. Pat. No.9,006,511, which is incorporated by reference herein in its entirety),in some embodiments referred to herein as HoH/LiK/λ^(−/−) mice, (2) arodent strain homozygous for a humanized immunoglobulin heavy chainlocus (supra), homozygous for an inactivated endogenous immunoglobulin λlight chain locus (supra), and hemizygous for an immunoglobulin κ lightchain locus having a first immunoglobulin κ light chain locus comprisinghuman Vλ and Jλ gene segments operably linked to a Cλ gene as describedherein and a second immunoglobulin κ light chain locus comprising humanVκ and Jκ gene segments operably linked to an endogenous mouse Cκ gene(see, e.g., U.S. Pat. Nos. 8,642,835 and 8,697,940, each of which isincorporated herein by reference in its entirety), in some embodimentsreferred to herein as HoH/KoK/LiK/λ^(−/−) mice. Alternatively, such micemaybe generated by introducing targeting vectors comprising engineeredloci into ES cells already comprising several engineered immunoglobulinloci. In some embodiments, the immunoglobulin heavy chain locus in saidrodents comprises a functional and expressed rodent Adam6 gene.

Specifically, LiK mice were bred with multiple engineered mouse strainsover multiple breedings to establish HoH/LiK/λ^(−/−) andHoH/KoK/LiK/λ^(−/−) mice.

Once established, various immune cell populations were characterized inthese humanized mice by flow cytometry. Briefly, spleens and femurs wereharvested from HoH/LiK/λ^(−/−) (n=3), HoH/KoK/LiK/λ^(−/−) (n=4) andVELOCIMMUNE® (“HoH/KoK”; n=3; see U.S. Pat. Nos. 8,642,835 and8,697,940, each of which is incorporated herein by reference in itsentirety) mice and prepared for flow cytometry analysis as describedabove. Representative results are set forth in FIGS. 8-11. Average lightchain expression (κ:λ) observed in splenocytes of engineered mousestrains tested was approximately as follows: HoH/LiK/λ^(−/−): 0:100,HoH/KoK/LiK/λ^(−/−): 40:60, HoH/KoK: 85:15.

Example 5. Production of Antibodies in Engineered Rodents

This example demonstrates production of antibodies in a rodent thatcomprises an engineered endogenous immunoglobulin κ light chain locus asdescribed above using an antigen of interest (e.g., a single-pass ormulti-pass membrane protein, etc.). The methods described in thisexample, or immunization methods well known in the art, can be used toimmunize rodents containing an engineered endogenous immunoglobulin κlight chain locus as described with various antigens (e.g.,polypeptides, etc.). Any genetically modified rodents described hereinabove, e.g., LiK mice-mice comprising an immunoglobulin κ light chainlocus comprising human Vλ and Jλ gene segments operably linked to a Cλgene (such as mice homozygous for the LiK locus); HoH/LiK/λ^(−/−)mice-mice comprising an LiK locus (such as mice homozygous for the LiKlocus) and also comprising humanized immunoglobulin heavy chain locus(see, e.g., U.S. Pat. Nos. 8,642,835 and 8,697,940, each of which isincorporated herein by reference in its entirety) and an inactivatedendogenous immunoglobulin λ light chain locus (see, e.g., U.S. Pat. No.9,006,511, which is incorporated by reference herein in its entirety);and HoH/KoK/LiK/λ^(−/−) mice-mice hemizygous for immunoglobulin κ lightchain locus having a first immunoglobulin κ light chain locus comprisingLiK and the second immunoglobulin κ light chain locus comprising humanVκ and Jκ gene segments operably linked to an endogenous mouse Cκ gene(see, e.g., U.S. Pat. Nos. 8,642,835 and 8,697,940), and also comprisinghumanized immunoglobulin heavy chain locus (see, e.g., U.S. Pat. Nos.8,642,835 and 8,697,940, each of which is incorporated herein byreference in its entirety) and an inactivated endogenous immunoglobulinλ light chain locus (see, e.g., U.S. Pat. No. 9,006,511, which isincorporated herein by reference), may be used for production ofantibodies after immunization with an antigen of interest. Such mice aresuitable for immunization and production of human antibodies and/orhuman antibody fragments.

LiK mice that further include the engineered immunoglobulin locidescribed above are challenged with an antigen of interest usingimmunization methods known in the art. The antibody immune response ismonitored by an ELISA immunoassay (i.e., serum titer). When a desiredimmune response is achieved, splenocytes (and/or other lymphatic tissue)are harvested and fused with mouse myeloma cells to preserve theirviability and form immortal hybridoma cell lines. Generated hybridomacell lines are screened (e.g., by an ELISA assay) and selected toidentify hybridoma cell lines that produce antigen-specific antibodies.Hybridomas may be further characterized for relative binding affinityand isotype as desired. Using this technique, and the immunogendescribed above, several antigen-specific chimeric antibodies (i.e.,antibodies possessing human variable domains and rodent constantdomains) are obtained.

DNA encoding the variable regions of heavy chain and light chains may beisolated or otherwise prepared, and may be linked to human heavy chainand light chain constant regions (e.g., of a desired isotype) for thepreparation of fully-human antibodies. Such fully-human antibodies(and/or heavy or light chains thereof) may be produced in a cell,typically a mammalian cell such as a CHO cell. Fully human antibodiesmay then be characterized for relative binding affinity and/orneutralizing activity of the antigen of interest.

DNA encoding antigen-specific chimeric antibodies produced by B cells ofthe engineered mice described and/or exemplified herein, and/or thevariable domains of light and/or heavy chains thereof, may be isolateddirectly from antigen-specific lymphocytes. For example, high affinitychimeric antibodies having a human variable region and a rodent constantregion may be isolated and characterized so that particular antibodies(and/or B cells that produce them) of interest are defined. To give buta few examples, assessed characteristics of such antibodies, and/orvariable and/or constant regions thereof, may be or include one or moreof affinity, selectivity, identity of epitope, etc.

Rodent constant regions are replaced with a desired human constantregion to generate fully-human antibodies. While the constant regionselected may vary according to specific use, high affinityantigen-binding and target specificity characteristics reside in thevariable region. Alternatively, when employing LiK mice containing ahuman Cλ2 gene in the place of a rodent Cκ gene as described herein, astep of replacing a rodent constant region in an antibody isolated froman immunized mouse is omitted. Antigen-specific antibodies are alsoisolated directly from antigen-positive B cells (from immunized mice)without fusion to myeloma cells, as described in, e.g., U.S. Pat. No.7,582,298, specifically incorporated herein by reference in itsentirety. Using this method, several fully human antigen-specificantibodies (i.e., antibodies possessing human variable domains and humanconstant domains) are made.

Example 6. Generation of Rodents Having an Engineered Light Chain Locusand Expressing Human Terminal Deoxynucleotidyl Transferase (TdT) GeneExample 6.1. Generation of Rodents Having an Engineered Light ChainLocus and Expressing Human TDT

This example illustrates the generation of mice whose germline genomecomprises an engineered immunoglobulin κ light chain locus as describedherein and further expressing human TdT. Mice expressing human TdT weremade as described in Example 1.1. of WO 2017/210586, incorporated hereinby reference in its entirety. Mice having a genome comprising both anengineered immunoglobulin κ light chain locus as described herein andfurther expressing human TdT were generated by multiple breedings toestablish cohorts of mouse strains containing both modifications.

Example 6.2. Phenotypic Assessment of Rodents Having an Engineered KappaLocus and Expressing Human TDT

Once established, immune cell populations were characterized in thesehumanized mice by flow cytometry. Briefly, spleens and femurs wereharvested from HoH/LiK/λ^(−/−)/TdT (n=4) and HoH/KoK/LiK/λ^(−/−)/TdT(n=6) mice and prepared for flow cytometry analysis as described above(see Example 3 above). Representative results are set forth in FIGS.14-17. Average light chain expression (κ:λ) observed in splenocytes ofengineered mouse strains tested was as follows: HoH/LiK/λ^(−/−)/TdT:0:100, HoH/KoK/LiK/λ^(−/−)/TdT: 45:55.

Example 6.3. Human Immunoglobulin Kappa Junctional Diversity andNon-Germline Additions in LiK Mice Comprising Human TdTS

As demonstrated in WO 2017/210586 (incorporated herein by reference inits entirety), mice comprising exogenously introduced TdT exhibitedincreases in both junctional diversity and non-germline nucleotideadditions (also “non-template nucleotide additions” as used herein) intheir light chains. The mice comprising HoH/LiK/λ^(−/−)/TdT andHoH/KoK/LiK/λ^(−/−)/TdT were assessed to determine their immunoglobulinrepertoire sequence diversity and presence of non-template nucleotideadditions in their CDR3 using Next Generation Sequencing technology.

Briefly, splenocytes were harvested from mice and B cells werepositively enriched from total splenocytes by anti-mouse CD19 magneticbeads and MACS columns (Miltenyi Biotech). Total RNA was isolated fromsplenic B cells using the RNeasy Plus kit (Qiagen).

Reverse transcription with an oligo-dT primer followed by gene specificPCR was performed to generate cDNA containing mouse Cλ1 sequence, usingSMARTer™ RACE cDNA Amplification Kit (Clontech). During reversetranscription, a specific DNA sequence (PIIA: 5′-CCCATGTACT CTGCGTTGATACCACTGCTT-3′, SEQ ID NO:71) was attached to the 3′ end of the newlysynthesized cDNAs. The cDNAs were purified by the NucleoSpin Gel and PCRClean-Up Kit (Clontech), then further amplified using a primer reversecompliment to PIIA (5′-AAGCAGTGGT ATCAACGCAG AGTACAT-3′, SEQ ID NO:72)paired with mouse Cλ1 specific primer (5′-CACCAGTGTG GCCTTGTTAG TCTC-3′,SEQ ID NO:73).

Purified amplicons were then amplified by PCR using a PIIA specificprimer (5′-GTGACTGGAG TTCAGACGTG TGCTCTTCCG ATCTAAGCAG TGGTATCAACGCAGAGT-3′, SEQ ID NO:74 and a nested mouse Cλ1 specific primer(5′-ACACTCTTTC CCTACACGAC GCTCTTCCGA TCTAAGGTGG AAACAGGGTG ACTGATG-3′,SEQ ID NO:75. PCR products between 450-690 bp were isolated andcollected by Pippin Prep (SAGE Science). These fragments were furtheramplified by PCR using following primers: 5′-AATGATACGG CGACCACCGAGATCTACACλ XXXXXACACT CTTTCCCTAC ACGACGCTCT TCCGATC-3′, SEQ ID NO:76 and5′-CAAGCAGAAG ACGGCATACG AGATXXXXXX GTGACTGGAG TTCAGACGTG TGCTCTTCCGATCT-3′, SEQ ID NO:77 (“XXXXXX” represents a 6 bp index sequence toenable multiplexing samples for sequencing). PCR products between 490bp-710 bp were isolated and collected by Pippin Prep, then quantified byqPCR using a KAPA Library Quantification Kit (KAPA Biosystems) beforeloading onto Miseq sequencer (Illumina) for sequencing (v3, 600-cycles).

For bioinformatic analysis, the resulting Illumina sequences weredemultiplexed and trimmed for quality. Overlapping paired-end reads werethen assembled and annotated using local installation of igblast (NCBI,v2.2.25+). Reads were aligned to human germline Vλ and Jλ segmentsdatabase and sorted for the best hit. A sequence was marked as ambiguousand removed from analysis when multiple best hits with identical scorewere detected. A set of in-house perl scripts was developed to analyzeresults.

Lambda light chains from splenic B cells from HoH/LiK/λ^(−/−)/TdT mice,and both lambda and kappa light chains from splenic B cells fromHoH/KoK/LiK/λ^(−/−)/TdT mice, were tested for an increase innon-template additions and junctional diversity at lambda and/or kappaloci. Light chains from HoH/LiK/λ^(−/−)/TdT and HoH/KoK/LiK/λ^(−/−)/TdTmice exhibited at least a 2 fold increase in junctional diversity asmeasured by number of unique CDR3/10,000 reads (data not shown). Inaddition, about 50% of light chains (lambda and/or kappa) from inHoH/LiK/λ^(−/−)/TdT and HoH/KoK/LiK/λ^(−/−)/TdT mice exhibitednon-template additions as compared to light chains from control micewithout TdT, which only exhibited about 10% non-template additions (datanot shown).

Example 7. Immunization of Engineered Rodents and Analysis of ImmuneResponse to Immunogens

This example illustrates immunization of LiK/VI-3 and LiK/VI-3/TdT mice,and the analysis of serum antibody responses to the immunogens. Briefly,(1) VI-3/TdT (e.g., a positive control for human kappa light chain,which also has endogenous mouse lambda light chain loci) and VI-3 micewith human lambda light chains, (2) LiK/VI-3 and (3) LiK/VI-3/TdT,respectively, were immunized with protein immunogens using standardprotocols and adjuvants. The mice were bled prior to the initiation ofimmunization and periodically bled following immunogen boosts.Anti-serum titers were assayed on respective antigens.

Antibody titers in serum against immunogens were determined using ELISA.Ninety six-well microtiter plates (Pierce) were coated with antigens at2 μg/ml in phosphate-buffered saline (PBS, Irvine Scientific) overnightat 4° C. Plates were washed with PBS containing 0.05% Tween-20 (PBS-T,Sigma-Aldrich) and blocked with 250 μl of 0.5% bovine serum albumin(BSA, Sigma-Aldrich) in PBS for 1 hour at room temperature. The plateswere washed with PBS-T. Pre-immune and immune anti-sera were seriallydiluted three-fold in 1% BSA-PBS and added to the plates for 1 hour atroom temperature. The plates were washed and goat anti-mouseIgG-Fc-Horse Radish Peroxidase (HRP) conjugated secondary antibodies(Jackson Immunoresearch), goat anti-mouse Kappa-HRP (SouthernBiotech) orgoat anti-mouse Lambda-HRP (SouthernBiotech) were added at 1:5000dilution to the plates and incubated for 1 hour at room temperature.Plates were washed and developed using TMB/H₂O₂ as substrate byincubating for 15-20 minutes. The reaction was stopped with acid andplates read on a spectrophotometer (Victor, Perkin Elmer) at 450 nm.Antibody titers were computed using Graphpad PRISM software. The titeris defined as interpolated serum dilution factor of which the bindingsignal is 2-fold over background.

The humoral immune responses in LiK/VI-3, LiK/VI-3/TdT, and VI-3/TdTmice were investigated following immunization with a protein immunogen.Antisera from mice immunized with protein show high titers on antigen inLiK/VI-3 and LiK/VI-3/TdT strains comparable to VI-3/TdT strain (FIG.18). High lambda titers were elicited in both LiK/VI-3 and LiK/VI-3/TdTmice. In VI-3/TdT strain mice, lambda titers were not observed in threemice, while low titers were observed in two mice, which corresponds tothe low usage of mouse lambda variables in this strain. As expected, nokappa titers were elicited in LiK/VI-3 and LiK/VI-3/TdT mice as theylack the kappa light chain, while the VI-3/TdT showed high kappa titers.FIG. 19 shows baseline titers (lowest serum dilution) were observed onirrelevant protein antigen for His tag in all three mice strains withanti-Fc and anti-kappa detection, while very low titers were observedwith anti-lambda detection in LiK/VI-3 and LiK/VI-3/TdT mice.

CERTAIN EMBODIMENTS Embodiment 1

A genetically modified rodent, whose germline genome comprises:

-   -   a first engineered endogenous immunoglobulin κ light chain locus        comprising:        -   (a) one or more human Vλ gene segments,        -   (b) one or more human Jλ gene segments, and        -   (c) a Cλ gene,    -   wherein the one or more human Vλ gene segments and the one or        more human Jλ gene segments are operably linked to the Cλ gene;        and

wherein the rodent lacks a rodent Cr gene at the first engineeredendogenous immunoglobulin κ light chain locus.

Embodiment 2

The genetically modified rodent of embodiment 1, wherein the rodent ishomozygous for the first engineered endogenous immunoglobulin κ lightchain locus.

Embodiment 3

The genetically modified rodent of embodiment 1, wherein the rodent isheterozygous for the first engineered endogenous immunoglobulin κ lightchain locus.

Embodiment 4

The genetically modified rodent of embodiment 3, wherein the germlinegenome of the rodent comprises a second engineered endogenousimmunoglobulin κ light chain locus comprising:

(a) one or more human Vκ gene segments, and

(b) one or more human Jκ gene segments,

wherein the one or more human Vκ gene segments and the one or more humanJκ gene segments are operably linked to a Cκ gene.

Embodiment 5

The genetically modified rodent of embodiment 4, wherein the Cκ gene atthe second engineered endogenous immunoglobulin κ light chain locus isan endogenous rodent Cκ gene.

Embodiment 6

The genetically modified rodent of any one of embodiments 1-5, whereinthe rodent lacks a rodent Cκ gene at the first engineered endogenousimmunoglobulin κ light chain locus.

Embodiment 7

A genetically modified rodent, whose germline genome comprises:

(a) a first engineered endogenous immunoglobulin κ light chain locuscomprising:

-   -   (i) one or more human Vλ gene segments,    -   (ii) one or more human Jλ gene segments, and    -   (iii) a Cλ gene,    -   wherein the one or more human Vλ gene segments and the one or        more human Jλ gene segments are operably linked to the Cλ gene,        and    -   wherein the rodent lacks a rodent Cκ gene at the first        engineered endogenous immunoglobulin κ light chain locus; and

(b) a second engineered endogenous immunoglobulin κ light chain locuscomprising:

-   -   (i) one or more human Vκ gene segments, and    -   (ii) one or more human Jκ gene segments,

wherein the one or more human Vκ gene segments and the one or more humanJκ gene segments are operably linked to a Cκ gene.

Embodiment 8

The genetically modified rodent of embodiment 7, wherein the Cκ gene atthe second engineered endogenous immunoglobulin κ light chain locus isan endogenous rodent Cκ gene.

Embodiment 9

The genetically modified rodent of any one of embodiments 1-8, whereinthe Cλ gene at the first engineered endogenous immunoglobulin κ lightchain locus comprises a rodent Cλ gene.

Embodiment 10

The genetically modified rodent of any one of embodiments 1-9, whereinthe first engineered endogenous immunoglobulin κ light chain locusfurther comprises:

(i) one or more human Vλ non-coding sequences, each of which is adjacentto at least one of the one or more human Vλ gene segments, wherein theone or more human Vλ non-coding sequences naturally appears adjacent toa human Vλ gene segment in an endogenous human immunoglobulin λ lightchain locus;

(ii) one or more human Jλ non-coding sequences, each of which isadjacent to at least one of the one or more human Jλ gene segments,wherein the one or more human Jλ non-coding sequences naturally appearsadjacent to a human Jλ gene segment in an endogenous humanimmunoglobulin λ light chain locus; or

(iii) any combination thereof.

Embodiment 11

The genetically modified rodent of any one of embodiments 1-10, wherein:

(i) the one or more human Vλ gene segments comprise Vλ5-52, Vλ1-51,Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37,Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14,Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, Vλ3-1, or any combinationthereof; and

(ii) the one or more human Jλ gene segments comprise Jλ1, Jλ2, Jλ3, Jλ6,Jλ7, or any combination thereof.

Embodiment 12

The genetically modified rodent of embodiment 11, wherein the firstengineered endogenous immunoglobulin κ light chain locus comprises:

(i) one or more human Vλ non-coding sequences, wherein each of the oneor more human Vλ non-coding sequences is adjacent to the Vλ5-52, Vλ1-51,Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37,Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14,Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, or Vλ3-1 in the firstengineered endogenous immunoglobulin κ light chain locus, and whereineach of the one or more human Vλ non-coding sequences naturally appearsadjacent to a Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44,Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22,Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8,Vλ4-3, or Vλ3-1 of an endogenous human immunoglobulin λ light chainlocus; and

(ii) one or more human Jλ non-coding sequences, wherein each of the oneor more human Jλ non-coding sequences is adjacent to the Jλ1, Jλ2, Jλ3,Jλ6 or Jλ7 in the first engineered endogenous immunoglobulin κ lightchain locus, and wherein each of the one or more human Jx non-codingsequences naturally appears adjacent to a Jλ1, Jλ2, Jλ3, Jλ6 or Jλ7 ofan endogenous human immunoglobulin λ light chain locus.

Embodiment 13

The genetically modified rodent of embodiment 11, wherein the firstengineered endogenous immunoglobulin κ light chain locus comprises:

(i) one or more human Vλ non-coding sequences, wherein each of the oneor more human Vλ non-coding sequences is adjacent to the Vλ5-52, Vλ1-51,Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37,Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16,Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3 or Vλ3-1 in thefirst engineered endogenous immunoglobulin κ light chain locus, andwherein each of the one or more human Vλ non-coding sequences naturallyappears adjacent to a Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45,Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23,Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10,Vλ3-9, Vλ2-8, Vλ4-3 or Vλ3-1 of an endogenous human immunoglobulin λlight chain locus; and

(ii) one or more human Jκ non-coding sequences, wherein each of the oneor more human Jκ non-coding sequences is adjacent to the Jλ1, Jλ2, Jλ3,Jλ6 or Jλ7 in the first engineered endogenous immunoglobulin κ lightchain locus, and wherein each of the one or more human Jκ non-codingsequences naturally appears adjacent to a Jκ1, Jκ2, Jκ3, Jκ4, or Jκ5 ofan endogenous human immunoglobulin κ light chain locus.

Embodiment 14

The genetically modified rodent of any one of embodiments 1-13, wherein:

(i) the one or more human Vλ gene segments comprise Vλ4-69, Vλ8-61,Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45,Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23,Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9,Vλ2-8, Vλ4-3, Vλ3-1, or any combination thereof; and

(ii) the one or more human Jλ gene segments comprise Jλ1, Jλ2, Jλ3, Jλ6,Jλ7, or any combination thereof.

Embodiment 15

The genetically modified rodent of embodiment 14, wherein the firstengineered endogenous immunoglobulin κ light chain locus comprises:

(i) one or more human Vλ non-coding sequences, wherein each of the oneor more human Vλ non-coding sequences is adjacent to the Vλ4-69, Vλ8-61,Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45,Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23,Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9,Vλ2-8, Vλ4-3, or Vλ3-1 in the first engineered endogenous immunoglobulinκ light chain locus, and wherein each of the one or more human Vλnon-coding sequences naturally appears adjacent to a Vλ4-69, Vλ8-61,Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45,Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23,Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9,Vλ2-8, Vλ4-3, or Vλ3-1 of an endogenous human immunoglobulin λ lightchain locus; and

(ii) one or more human Jλ non-coding sequences, wherein each of the oneor more human Jλ non-coding sequences is adjacent to the Jλ1, Jλ2, Jλ3,Jλ6 or Jλ7 in the first engineered endogenous immunoglobulin κ lightchain locus, and wherein each of the one or more human Jλ non-codingsequences naturally appears adjacent to a Jλ1, Jλ2, Jλ3, Jλ6 or Jλ7 ofan endogenous human immunoglobulin λ light chain locus.

Embodiment 16

The genetically modified rodent of embodiment 14, wherein the firstengineered endogenous immunoglobulin κ light chain locus comprises:

(i) one or more human Vλ non-coding sequences, wherein each of the oneor more human Vλ non-coding sequences is adjacent to the Vλ4-69, Vλ8-61,Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45,Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23,Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10,Vλ3-9, Vλ2-8, Vλ4-3 or Vλ3-1 in the first engineered endogenousimmunoglobulin κ light chain locus, and wherein each of the one or morehuman Vλ non-coding sequences naturally appears adjacent to a Vλ4-69,Vλ8-61, Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46,Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25,Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11,Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3 or Vλ3-1 of an endogenous humanimmunoglobulin λ light chain locus; and

(ii) one or more human Jκ non-coding sequences, wherein each of the oneor more human Jκ non-coding sequences is adjacent to the Jλ1, Jλ2, Jλ3,Jλ6 or Jλ7 in the first engineered endogenous immunoglobulin κ lightchain locus, and wherein each of the one or more human Jκ non-codingsequences naturally appears adjacent to a Jκ1, Jκ2, Jκ3, Jκ4, or Jκ5 ofan endogenous human immunoglobulin κ light chain locus.

Embodiment 17

A genetically modified rodent, whose germline genome comprises:

a first engineered endogenous immunoglobulin κ light chain locuscomprising:

-   -   (i) one or more human Vλ gene segments, wherein the one or more        human Vλ gene segments comprise Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47,        Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36,        Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16,        Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, Vλ3-1, or        any combination thereof,    -   (ii) one or more human Jλ gene segments, wherein the one or more        human Jλ gene segments comprise Jλ1, Jλ2, Jλ3, Jλ6, Jλ7, or any        combination thereof,    -   (iii) a rodent Cλ gene,    -   (iv) one or more human Vλ non-coding sequences, wherein each of        the one or more human Vλ non-coding sequences is adjacent to the        Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43,        Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22,        Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10,        Vλ3-9, Vλ2-8, Vλ4-3 or Vλ3-1 in the engineered endogenous        immunoglobulin κ light chain locus, and wherein each of the one        or more human Vλ non-coding sequences naturally appears adjacent        to a Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44,        Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23,        Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11,        Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3 or Vλ3-1 of an endogenous human        immunoglobulin λ light chain locus,    -   (v) one or more human Jκ non-coding sequences, wherein each of        the one or more human Jκ non-coding sequences is adjacent to the        Jλ1, Jλ2, Jλ3, Jλ6, or Jλ7 in the engineered endogenous        immunoglobulin κ light chain locus, and wherein each of the one        or more human Jκ non-coding sequences naturally appears adjacent        to a Jκ1, Jκ2, Jκ3, Jκ4, or Jκ5 of an endogenous human        immunoglobulin κ light chain locus, and    -   (vi) a human κ light chain non-coding sequence between the one        or more human Vλ gene segments and the one or more human Jλ gene        segments that has a sequence that naturally appears between a        human Vκ4-1 gene segment and a human Jκ1 gene segment in an        endogenous human immunoglobulin κ light chain locus,

wherein the one or more human Vλ gene segments, the one or more human Jλgene segments, and the rodent Cλ gene are operably linked to each other,and

wherein the rodent Cλ gene is in place of a rodent Cκ gene of theendogenous immunoglobulin κ light chain locus.

Embodiment 18

The genetically modified rodent of embodiment 17, wherein the germlinegenome of the rodent comprises a second engineered endogenousimmunoglobulin κ light chain locus comprising:

(a) one or more human Vκ gene segments, and

(b) one or more human Jκ gene segments,

wherein the one or more human Vκ gene segments and the one or more humanJκ gene segments are operably linked to a Cκ gene.

Embodiment 19

The genetically modified rodent of embodiment 18, wherein the Cκ gene atthe second engineered endogenous immunoglobulin κ light chain locus isan endogenous rodent Cκ gene.

Embodiment 20

The genetically modified rodent of any one of embodiments 1-19, whereinthe germline genome of the rodent further comprises:

an engineered endogenous immunoglobulin heavy chain locus, comprising:

-   -   (a) one or more human V_(H) gene segments,    -   (b) one or more human D_(H) gene segments, and    -   (c) one or more human J_(H) gene segments,

wherein the one or more human V_(H) gene segments, the one or more humanD_(H) gene segments, and the one or more human J_(H) gene segments areoperably linked to one or more rodent immunoglobulin heavy chainconstant region genes at the engineered endogenous immunoglobulin heavychain locus.

Embodiment 21

A genetically modified rodent whose germline genome comprises:

(a) an engineered endogenous immunoglobulin heavy chain locus comprisingone or more human V_(H) gene segments, one or more human D_(H) genesegments, and one or more human J_(H) gene segments operably linked toone or more endogenous immunoglobulin heavy chain constant region genessuch that the rodent expresses immunoglobulin heavy chains that comprisea human heavy chain variable domain sequence and a rodent heavy chainconstant domain sequence, wherein the germline genome is homozygous forthe engineered endogenous immunoglobulin heavy chain locus;

(b) a first engineered endogenous immunoglobulin κ light chain locuscomprising:

-   -   (i) one or more human Vλ gene segments, wherein the one or more        human Vλ gene segments comprise Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47,        Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36,        Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16,        Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, Vλ3-1, or        any combination thereof,    -   (ii) one or more human Jλ gene segments, wherein the one or more        human Jλ gene segments comprise Jλ1, Jλ2, Jλ3, Jλ6, Jλ7, or any        combination thereof,    -   (iii) a rodent Cλ gene,    -   (iv) one or more human Vλ non-coding sequences, wherein each of        the one or more human Vλ non-coding sequences is adjacent to the        Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43,        Vλ1-40, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18,        Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3 or        Vλ3-1 in the second engineered endogenous immunoglobulin κ light        chain locus, and wherein each of the one or more human Vλ        non-coding sequences naturally appears adjacent to a Vλ5-52,        Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40,        Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16,        Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3 or Vλ3-1 of        an endogenous human immunoglobulin λ light chain locus,    -   (v) one or more human Jκ non-coding sequences, wherein each of        the one or more human Jκ non-coding sequences is adjacent to the        Jλ1, Jλ2, Jλ3, Jλ6 or Jλ7 in the second engineered endogenous        immunoglobulin κ light chain locus, and wherein each of the one        or more human Jκ non-coding sequences naturally appears adjacent        to a Jκ1, Jκ2, Jκ3, Jκ4, or Jκ5 of an endogenous human        immunoglobulin κ light chain locus, and    -   (vi) a human κ light chain non-coding sequence between the one        or more human Vλ gene segments and the one or more human Jλ gene        segments that has a sequence that naturally appears between a        human Vκ4-1 gene segment and a human Jκ1 gene segment in an        endogenous human immunoglobulin κ light chain locus,

wherein the one or more human Vλ gene segments, the one or more human Jλgene segments, and the rodent Cλ gene are operably linked to each other,

wherein the rodent Cλ gene is in place of a rodent Cκ gene at the secondendogenous immunoglobulin κ light chain locus; and

(c) a second engineered endogenous immunoglobulin κ light chain locuscomprising one or more human Vκ gene segments and one or more Jκ genesegments operably linked to an endogenous rodent Cκ region gene suchthat the rodent expresses immunoglobulin light chains that comprise ahuman κ light chain variable domain sequence and a rodent κ light chainconstant domain sequence,

wherein the rodent expresses immunoglobulin light chains that comprise ahuman λ light chain variable domain sequence and a rodent λ light chainconstant domain sequence.

Embodiment 22

The genetically modified rodent of embodiment 20 or 21, wherein the oneor more human V_(H) gene segments, one or more human D_(H) genesegments, and one or more human J_(H) gene segments are in place of oneor more rodent V_(H) gene segments, one or more rodent D_(H) genesegments, one or more rodent J_(H) gene segments, or a combinationthereof.

Embodiment 23

The genetically modified rodent of any one of embodiments 20-22, whereinthe one or more human V_(H) gene segments, one or more human D_(H) genesegments, and one or more human J_(H) gene segments replace one or morerodent V_(H) gene segments, one or more rodent D_(H) gene segments, oneor more rodent J_(H) gene segments, or any combination thereof.

Embodiment 24

The genetically modified rodent of any one of embodiments 20-23, whereinthe engineered endogenous immunoglobulin heavy chain locus furthercomprises:

(i) one or more human V_(H) non-coding sequences, each of which isadjacent to at least one of the one or more human V_(H) gene segments,wherein each of the one or more V_(H) non-coding sequences naturallyappears adjacent to a human V_(H) gene segment in an endogenous humanimmunoglobulin heavy chain locus;

(ii) one or more human D_(H) non-coding sequences, each of which isadjacent to at least one of the one or more human D_(H) gene segments,wherein each of the one or more D_(H) non-coding sequences naturallyappears adjacent to a human D_(H) gene segment in an endogenous humanimmunoglobulin heavy chain locus;

(iii) one or more human J_(H) non-coding sequences, each of which isadjacent to at least one of the one or more human J_(H) gene segments,wherein each of the one or more J_(H) non-coding sequences naturallyappears adjacent to a human J_(H) gene segment in an endogenous humanimmunoglobulin heavy chain locus; or

(iv) any combination thereof.

Embodiment 25

The genetically modified rodent of any one of embodiments 20-24, whereinthe one or more rodent immunoglobulin heavy chain constant region genesare one or more endogenous rodent immunoglobulin heavy chain constantregion genes.

Embodiment 26

The genetically modified rodent of any one of embodiments 20-25,wherein:

(i) the one or more human V_(H) gene segments comprise V_(H)3-74,V_(H)3-73, V_(H)3-72, V_(H)2-70, V_(H)1-69, V_(H)3-66, V_(H)3-64,V_(H)4-61, V_(H)4-59, V_(H)1-58, V_(H)3-53, V_(H)5-51, V_(H)3-49,V_(H)3-48, V_(H)1-46, V_(H)1-45, V_(H)3-43, V_(H)4-39, V_(H)4-34,V_(H)3-33, V_(H)4-31, V_(H)3-30, V_(H)4-28, V_(H)2-26, V_(H)1-24,V_(H)3-23, V_(H)3-21, V_(H)3-20, V_(H)1-18, V_(H)3-15, V_(H)3-13,V_(H)3-11, V_(H)3-9, V_(H)1-8, V_(H)3-7, V_(H)2-5, V_(H)7-4-1, V_(H)4-4,V_(H)1-3, V_(H)1-2, V_(H)6-1, or any combination thereof,

(ii) the one or more human D_(H) gene segments comprise D_(H)1-1,D_(H)2-2, D_(H)3-3, D_(H)4-4, D_(H)5-5, D_(H)6-6, D_(H)1-7, D_(H)2-8,D_(H)3-9, D_(H)3-10, D_(H)5-12, D_(H)6-13, D_(H)2-15, D_(H)3-16,D_(H)4-17, D_(H)6-19, D_(H)1-20, D_(H)2-21, D_(H)3-22, D_(H)6-25,D_(H)1-26, D_(H)7-27, or any combination thereof, and

(iii) the one or more human J_(H) gene segments comprise J_(H)1, J_(H)2,J_(H)3, J_(H)4, J_(H)5, J_(H)6, or any combination thereof.

Embodiment 27

The genetically modified rodent of any one of embodiments 20-26, whereinthe engineered endogenous immunoglobulin heavy chain locus lacks afunctional endogenous rodent Adam6 gene.

Embodiment 28

The genetically modified rodent of any one of embodiments 20-27, whereinthe germline genome further comprises one or more nucleotide sequencesencoding one or more rodent ADAM6 polypeptides, functional orthologs,functional homologs, or functional fragments thereof.

Embodiment 29

The genetically modified rodent of embodiment 28, wherein the one ormore rodent ADAM6 polypeptides, functional orthologs, functionalhomologs, or functional fragments thereof are expressed.

Embodiment 30

The genetically modified rodent of embodiment 28 or 29, wherein the oneor more nucleotide sequences encoding one or more rodent ADAM6polypeptides, functional orthologs, functional homologs, or functionalfragments thereof are included on the same chromosome as the engineeredendogenous immunoglobulin heavy chain locus.

Embodiment 31

The genetically modified rodent of any one of embodiments 28-30, whereinthe one or more nucleotide sequences encoding one or more rodent ADAM6polypeptides, functional orthologs, functional homologs, or functionalfragments thereof are included in the engineered endogenousimmunoglobulin heavy chain locus.

Embodiment 32

The genetically modified rodent of any one of embodiments 28-31, whereinthe one or more nucleotide sequences encoding one or more rodent ADAM6polypeptides, functional orthologs, functional homologs, or functionalfragments thereof are in place of a human Adam6 pseudogene.

Embodiment 33

The genetically modified rodent of any one of embodiments 28-32, whereinthe one or more nucleotide sequences encoding one or more rodent ADAM6polypeptides, functional orthologs, functional homologs, or functionalfragments thereof replace a human Adam6 pseudogene.

Embodiment 34

The genetically modified rodent of any one of embodiments 28-33, whereinthe one or more nucleotide sequences encoding one or more rodent ADAM6polypeptides, functional orthologs, functional homologs, or functionalfragments thereof are between a first human V_(H) gene segment and asecond human V_(H) gene segment.

Embodiment 35

The genetically modified rodent of embodiment 34, wherein the firsthuman V_(H) gene segment is V_(H)1-2 and the second human V_(H) genesegment is V_(H)6-1.

Embodiment 36

The genetically modified rodent of any one of embodiments 28-31, whereinthe one or more nucleotide sequences encoding one or more rodent ADAM6polypeptides, functional orthologs, functional homologs, or functionalfragments thereof are between a human V_(H) gene segment and a humanD_(H) gene segment.

Embodiment 37

The genetically modified rodent of any one embodiments 20, and 22-36,wherein the rodent is homozygous for the engineered endogenousimmunoglobulin heavy chain locus.

Embodiment 38

The genetically modified rodent of any one of embodiments 9-37, whereinthe rodent Cλ gene has a sequence that is at least 80% identical to amouse Cλ1, mouse Cλ2 or a mouse Cλ3 gene.

Embodiment 39

The genetically modified rodent of any one of embodiments 9-38, whereinthe rodent Cλ gene comprises a mouse Cλ1 gene.

Embodiment 40

The genetically modified rodent of any one of embodiments 9-39, whereinthe rodent Cλ gene comprises a rat Cλ gene.

Embodiment 41

The genetically modified rodent of embodiment 40, wherein the rat Cλgene has a sequence that is at least 80% identical to a rat Cλ1, ratCλ2, rat Cλ3 or a rat Cλ4 gene.

Embodiment 42

The genetically modified rodent of any one of embodiments 1-41, whereinthe one or more human Vλ gene segments and the one or more human Jλ genesegments are in place of one or more rodent Vκ gene segments, one ormore rodent Jκ gene segments, or any combination thereof.

Embodiment 43

The genetically modified rodent of any one of embodiments 1-42, whereinthe one or more human Vλ gene segments and the one or more human Jλ genesegments replace one or more rodent Vκ gene segments, one or more rodentJκ gene segments, or any combination thereof.

Embodiment 44

The genetically modified rodent of any one of embodiments 1-43, whereinthe first engineered endogenous immunoglobulin κ light chain locusfurther comprises a κ light chain non-coding sequence between the one ormore human Vλ gene segments and the one or more human Jλ gene segments.

Embodiment 45

The genetically modified rodent of embodiment 44, wherein the κ lightchain non-coding sequence is a human κ light chain non-coding sequence.

Embodiment 46

The genetically modified rodent of embodiment 45, wherein the human Klight chain non-coding sequence has a sequence that naturally appearsbetween a human Vκ4-1 gene segment and a human Jκ1 gene segment in anendogenous human immunoglobulin κ light chain locus.

Embodiment 47

The genetically modified rodent of any one of embodiments 1-46, furthercomprising an inactivated endogenous immunoglobulin λ light chain locus.

Embodiment 48

The genetically modified rodent of embodiment 47, wherein the rodent isheterozygous for the inactivated endogenous immunoglobulin λ light chainlocus.

Embodiment 49

The genetically modified rodent of embodiment 47, wherein the rodent ishomozygous for the inactivated endogenous immunoglobulin λ light chainlocus.

Embodiment 50

The genetically modified rodent of any one of embodiments 1-49, whereinthe endogenous Vλ gene segments, the endogenous Jλ gene segments, andthe endogenous Cλ genes are deleted in whole or in part.

Embodiment 51

The genetically modified rodent of any one of embodiments 1-50, whereinthe rodent does not detectably express endogenous immunoglobulin λ lightchain variable domains.

Embodiment 52

The genetically modified rodent of any one of embodiments 1-51, whereinthe rodent does not detectably express endogenous immunoglobulin κ lightchain variable domains.

Embodiment 53

The genetically modified rodent of any one of embodiments 1-52, whereinthe rodent comprises a population of B cells that express antibodies,including immunoglobulin λ light chains that each include a humanimmunoglobulin λ light chain variable domain.

Embodiment 54

The genetically modified rodent of embodiment 53, wherein the humanimmunoglobulin λ light chain variable domain is encoded by a rearrangedhuman immunoglobulin λ light chain variable region sequence comprising(i) one of the one or more human Vλ gene segments or a somaticallyhypermutated variant thereof, and (ii) one of the one or more human Jλgene segments or a somatically hypermutated variant thereof.

Embodiment 55

The genetically modified rodent of any one of embodiments 1-54, whereinthe germline genome of the rodent further comprises a nucleic acidsequence encoding an exogenous terminal deoxynucleotidyltransferase(TdT) operably linked to a transcriptional control element.

Embodiment 56

The genetically modified rodent of embodiment 55, wherein the TdT is ashort isoform of TdT (TdTS).

Embodiment 57

The genetically modified rodent of embodiment 55 or 56, wherein thetranscriptional control element comprises a RAG1 transcriptional controlelement, a RAG2 transcriptional control element, an immunoglobulin heavychain transcriptional control element, an immunoglobulin κ light chaintranscriptional control element, an immunoglobulin λ light chaintranscriptional control element, or any combination thereof.

Embodiment 58

The genetically modified rodent of any one of embodiments 55-57, whereinthe nucleic acid sequence encoding an exogenous TdT is in the germlinegenome at an immunoglobulin κ light chain locus, an immunoglobulin λlight chain locus, an immunoglobulin heavy chain locus, a RAG1 locus, ora RAG2 locus.

Embodiment 59

A genetically modified rodent whose germline genome comprises

(a) a nucleic acid sequence encoding an exogenous terminaldeoxynucleotidyltransferase (TdT) operably linked to a transcriptionalcontrol element;

(b) an engineered endogenous immunoglobulin heavy chain locus comprisingone or more human V_(H) gene segments, one or more human D_(H) genesegments and one or more human J_(H) gene segments operably linked toone or more endogenous immunoglobulin heavy chain constant region genessuch that the rodent expresses immunoglobulin heavy chains that eachcomprise a human heavy chain variable domain sequence and a rodent heavychain constant domain sequence, wherein the germline genome ishomozygous for the engineered endogenous immunoglobulin heavy chainlocus; and

(c) an engineered endogenous immunoglobulin κ light chain locuscomprising:

-   -   (i) one or more human Vλ gene segments, wherein the one or more        human Vλ gene segments comprise Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47,        Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36,        Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16,        Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, Vλ3-1 or        any combination thereof,    -   (ii) one or more human Jλ gene segments, wherein the one or more        human Jλ gene segments comprise Jλ1, Jλ2, Jλ3, Jλ6, Jλ7 or any        combination thereof,    -   (iii) a rodent Cλ gene,    -   (iv) one or more human Vλ non-coding sequences, wherein each of        the one or more human Vλ non-coding sequences is adjacent to the        Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43,        Vλ1-40, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18,        Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3 or        Vλ3-1 in the engineered endogenous immunoglobulin κ light chain        locus, and wherein each of the one or more human Vλ non-coding        sequences naturally appears adjacent to a Vλ5-52, Vλ1-51,        Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ3-27,        Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14,        Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3 or Vλ3-1 of an        endogenous human immunoglobulin λ light chain locus,    -   (v) one or more human Jκ non-coding sequences, wherein each of        the one or more human Jκ non-coding sequences is adjacent to the        Jλ1, Jλ2, Jλ3, Jλ6 and Jλ7 in the engineered endogenous        immunoglobulin κ light chain locus, and wherein each of the one        or more human Jκ non-coding sequences naturally appears adjacent        to a Jκ1, Jκ2, Jκ3, Jκ4, or Jκ5 of an endogenous human        immunoglobulin κ light chain locus, and    -   (vi) a human κ light chain non-coding sequence between the one        or more human Vλ gene segments and the one or more human Jλ gene        segments that comprises a sequence that naturally appears        between a human Vκ4-1 gene segment and a human Jκ1 gene segment        in an endogenous human immunoglobulin κ light chain locus,

wherein the one or more human Vλ gene segments, the one or more human Jλgene segments, and the rodent Cλ gene are operably linked to each other,

wherein the rodent Cλ gene is in place of a rodent Cκ gene of theendogenous immunoglobulin κ light chain locus,

wherein the germline genome is homozygous for the engineered endogenousimmunoglobulin κ light chain locus, and

wherein the rodent expresses immunoglobulin light chains that comprise ahuman λ light chain variable domain sequence fused to a rodent λ lightchain constant domain sequence.

Embodiment 60

The genetically modified rodent of any one of embodiments 1-59, whereinthe rodent is a rat or a mouse.

Embodiment 61

An isolated rodent cell obtained from a rodent of any one of embodiments1-60.

Embodiment 62

An immortalized cell generated from the isolated rodent cell ofembodiment 61.

Embodiment 63

The isolated rodent cell of 61, wherein the rodent cell is a rodentembryonic stem (ES) cell.

Embodiment 64

A rodent embryo generated from the rodent ES cell of embodiment 63.

Embodiment 65

Use of a rodent of any one of embodiments 1-60 for making an antibody.

Embodiment 66

Use of a rodent of any one of embodiments 1-60 for making a light chainvariable region sequence.

Embodiment 67

Use of a rodent of any one of embodiments 1-60 for making a light chainvariable domain sequence.

Embodiment 68

An isolated B cell obtained from a rodent of any one embodiments 1-60,wherein the genome of the B cell comprises:

(a) a rearranged human lambda light chain variable region sequenceoperably linked to a lambda light chain variable region sequence,wherein the rearranged human lambda light chain variable region sequencecomprises:

-   -   (i) one of the one or more human Vλ gene segments or a        somatically hypermuted variant thereof, and    -   (ii) one of the one or more human Jλ gene segments or a        somatically hypermuted variant thereof.

Embodiment 69

The isolated B cell of embodiment 68, further comprising:

(b) a rearranged human heavy chain variable region sequence operablylinked to a rodent heavy chain variable region sequence, wherein therearranged human heavy chain variable region sequence comprises:

-   -   (i) one of the one or more human V_(H) gene segments or a        somatically hypermuted variant thereof,    -   (ii) one of the one or more human D_(H) gene segments or a        somatically hypermuted variant thereof, and    -   (iii) one of the one or more human J_(H) gene segments or a        somatically hypermuted variant thereof.

Embodiment 70

An antibody prepared by a method comprising the steps of:

(a) providing a rodent of any one of embodiments 1-60;

(b) immunizing the rodent with an antigen of interest;

(c) maintaining the rodent under conditions sufficient for the rodent toproduce an immune response to the antigen of interest; and

(d) recovering from the rodent:

-   -   (i) an antibody that binds the antigen of interest,    -   (ii) a nucleotide that encodes a human light or heavy chain        variable domain, a light chain, or a heavy chain of an antibody        that binds the antigen of interest, or    -   (iii) a cell that expresses an antibody that binds the antigen        of interest,

wherein an antibody of (d) includes human heavy chain variable and humanλ light chain variable domains.

Embodiment 71

An isolated rodent cell, whose genome comprises:

-   -   a first engineered endogenous immunoglobulin κ light chain locus        comprising:        -   (a) one or more human Vλ gene segments,        -   (b) one or more human Jλ gene segments, and        -   (c) a Cλ gene,    -   wherein the one or more human Vλ gene segments and the one or        more human Jλ gene segments are operably linked to the Cλ gene,        and

wherein the isolated rodent cell lacks a rodent Cκ gene at the firstengineered endogenous immunoglobulin κ light chain locus.

Embodiment 72

The isolated rodent cell of embodiment 71, wherein the isolated rodentcell is homozygous for the first engineered endogenous immunoglobulin κlight chain locus.

Embodiment 73

The isolated rodent cell of embodiment 71, wherein the isolated rodentcell is heterozygous for the first engineered endogenous immunoglobulinκ light chain locus.

Embodiment 74

The isolated rodent cell of embodiment 73, wherein the genome of theisolated rodent cell comprises a second engineered endogenousimmunoglobulin κ light chain locus comprising:

(a) one or more human Vκ gene segments, and

(b) one or more human Jκ gene segments,

wherein the one or more human Vκ gene segments and the one or more humanJκ gene segments are operably linked to a Cκ gene.

Embodiment 75

The isolated rodent cell of embodiment 74, wherein the Cκ gene at thesecond engineered endogenous immunoglobulin κ light chain locus is anendogenous rodent Cκ gene.

Embodiment 76

The isolated rodent cell of any one of embodiments 71-75, wherein theisolated rodent cell lacks a rodent Cκ gene at the first engineeredendogenous immunoglobulin κ light chain locus.

Embodiment 77

The isolated rodent cell of any one of embodiments 71-76, wherein the Cλgene at the first engineered endogenous immunoglobulin κ light chainlocus comprises a rodent Cλ gene.

Embodiment 78

The isolated rodent cell of any one of embodiments 71-77, wherein thefirst engineered endogenous immunoglobulin κ light chain locus furthercomprises:

(i) one or more human Vλ non-coding sequences, each of which is adjacentto at least one of the one or more human Vλ gene segments, wherein theone or more human Vλ non-coding sequences naturally appears adjacent toa human Vλ gene segment in an endogenous human immunoglobulin λ lightchain locus;

(ii) one or more human Jλ non-coding sequences, each of which isadjacent to at least one of the one or more human Jλ gene segments,wherein the one or more human Jλ non-coding sequences naturally appearsadjacent to a human Jλ gene segment in an endogenous humanimmunoglobulin λ light chain locus; or

(iii) any combination thereof.

Embodiment 79

The isolated rodent cell of any one of embodiments 71-78, wherein:

(i) the one or more human Vλ gene segments comprise Vλ5-52, Vλ1-51,Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37,Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14,Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, Vλ3-1, or any combinationthereof; and

(ii) the one or more human Jλ gene segments comprise Jλ1, Jλ2, Jλ3, Jλ6,Jλ7, or any combination thereof.

Embodiment 80

The isolated rodent cell of embodiment 79, wherein the first engineeredendogenous immunoglobulin κ light chain locus comprises:

(i) one or more human Vλ non-coding sequences, wherein each of the oneor more human Vλ non-coding sequences is adjacent to the Vλ5-52, Vλ1-51,Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37,Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14,Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, or Vλ3-1 in the firstengineered endogenous immunoglobulin κ light chain locus, and whereineach of the one or more human Vλ non-coding sequences naturally appearsadjacent to a Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44,Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22,Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8,Vλ4-3, or Vλ3-1 of an endogenous human immunoglobulin λ light chainlocus; and

(ii) one or more human Jλ non-coding sequences, wherein each of the oneor more human Jλ non-coding sequences is adjacent to the Jλ1, Jλ2, Jλ3,Jλ6 or Jλ7 in the first engineered endogenous immunoglobulin κ lightchain locus, and wherein each of the one or more human Jx non-codingsequences naturally appears adjacent to a Jλ1, Jλ2, Jλ3, Jλ6 or Jλ7 ofan endogenous human immunoglobulin λ light chain locus.

Embodiment 81

The isolated rodent cell of embodiment 79, wherein the first engineeredendogenous immunoglobulin κ light chain locus comprises:

(i) one or more human Vλ non-coding sequences, wherein each of the oneor more human Vλ non-coding sequences is adjacent to the Vλ5-52, Vλ1-51,Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37,Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16,Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3 or Vλ3-1 in thefirst engineered endogenous immunoglobulin κ light chain locus, andwherein each of the one or more human Vλ non-coding sequences naturallyappears adjacent to a Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45,Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23,Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10,Vλ3-9, Vλ2-8, Vλ4-3 or Vλ3-1 of an endogenous human immunoglobulin λlight chain locus,

(ii) one or more human Jκ non-coding sequences, wherein each of the oneor more human Jκ non-coding sequences is adjacent to the Jλ1, Jλ2, Jλ3,Jλ6 or Jλ7 in the first engineered endogenous immunoglobulin κ lightchain locus, and wherein each of the one or more human Jκ non-codingsequences naturally appears adjacent to a Jκ1, Jκ2, Jκ3, Jκ4, or Jκ5 ofan endogenous human immunoglobulin κ light chain locus.

Embodiment 82

The isolated rodent cell of any one of embodiments 71-81, wherein:

(a) the one or more human Vλ gene segments comprise Vλ4-69, Vλ8-61,Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45,Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23,Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9,Vλ2-8, Vλ4-3, Vλ3-1, or any combination thereof; and

(b) the one or more human Jλ gene segments comprise Jλ1, Jλ2, Jλ3, Jλ6,Jλ7, or any combination thereof.

Embodiment 83

The isolated rodent cell of embodiment 82, wherein the first engineeredendogenous immunoglobulin κ light chain locus comprises:

(i) one or more human Vλ non-coding sequences, wherein each of the oneor more human Vλ non-coding sequences is adjacent to the Vλ4-69, Vλ8-61,Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45,Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23,Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9,Vλ2-8, Vλ4-3, or Vλ3-1 in the first engineered endogenous immunoglobulinκ light chain locus, and wherein each of the one or more human Vλnon-coding sequences naturally appears adjacent to a Vλ4-69, Vλ8-61,Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45,Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23,Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9,Vλ2-8, Vλ4-3, or Vλ3-1 of an endogenous human immunoglobulin λ lightchain locus; and

(ii) one or more human Jλ non-coding sequences, wherein each of the oneor more human Jλ non-coding sequences is adjacent to the Jλ1, Jλ2, Jλ3,Jλ6 or Jλ7 in the first engineered endogenous immunoglobulin κ lightchain locus, and wherein each of the one or more human Jλ non-codingsequences naturally appears adjacent to a Jλ1, Jλ2, Jλ3, Jλ6 or Jλ7 ofan endogenous human immunoglobulin λ light chain locus.

Embodiment 84

The isolated rodent cell of embodiment 82, wherein the first engineeredendogenous immunoglobulin κ light chain locus comprises:

(i) one or more human Vλ non-coding sequences, wherein each of the oneor more human Vλ non-coding sequences is adjacent to the Vλ4-69, Vλ8-61,Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45,Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23,Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10,Vλ3-9, Vλ2-8, Vλ4-3 or Vλ3-1 in the first engineered endogenousimmunoglobulin κ light chain locus, and wherein each of the one or morehuman Vλ non-coding sequences naturally appears adjacent to a Vλ4-69,Vλ8-61, Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46,Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25,Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11,Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3 or Vλ3-1 of an endogenous humanimmunoglobulin λ light chain locus; and

(ii) one or more human Jκ non-coding sequences, wherein each of the oneor more human Jκ non-coding sequences is adjacent to the Jλ1, Jλ2, Jλ3,Jλ6 or Jλ7 in the first engineered endogenous immunoglobulin κ lightchain locus, and wherein each of the one or more human Jκ non-codingsequences naturally appears adjacent to a Jκ1, Jκ2, Jκ3, Jκ4, or Jκ5 ofan endogenous human immunoglobulin κ light chain locus.

Embodiment 85

The isolated rodent cell of any one of embodiments 71-84, wherein thegenome of the rodent cell further comprises:

an engineered endogenous immunoglobulin heavy chain locus, comprising:

-   -   (a) one or more human V_(H) gene segments,    -   (b) one or more human D_(H) gene segments, and    -   (c) one or more human J_(H) gene segments,

wherein the one or more human V_(H) gene segments, the one or more humanD_(H) gene segments, and the one or more human J_(H) gene segments areoperably linked to one or more rodent immunoglobulin heavy chainconstant region genes at the engineered endogenous immunoglobulin heavychain locus.

Embodiment 86

The isolated rodent cell of embodiment 85, wherein the one or more humanV_(H) gene segments, one or more human D_(H) gene segments, and one ormore human J_(H) gene segments are in place of one or more rodent V_(H)gene segments, one or more rodent D_(H) gene segments, one or morerodent J_(H) gene segments, or a combination thereof.

Embodiment 87

The isolated rodent cell of embodiment 85 or 86, wherein the one or morehuman V_(H) gene segments, one or more human D_(H) gene segments, andone or more human J_(H) gene segments replace one or more rodent V_(H)gene segments, one or more rodent D_(H) gene segments, one or morerodent J_(H) gene segments, or any combination thereof.

Embodiment 88

The isolated rodent cell of any one of embodiments 85-87, wherein theengineered endogenous immunoglobulin heavy chain locus furthercomprises:

(i) one or more human V_(H) non-coding sequences, each of which isadjacent to at least one of the one or more human V_(H) gene segments,wherein each of the one or more V_(H) non-coding sequences naturallyappears adjacent to a human V_(H) gene segment in an endogenous humanimmunoglobulin heavy chain locus;

(ii) one or more human D_(H) non-coding sequences, each of which isadjacent to at least one of the one or more human D_(H) gene segments,wherein each of the one or more D_(H) non-coding sequences naturallyappears adjacent to a human D_(H) gene segment in an endogenous humanimmunoglobulin heavy chain locus;

(iii) one or more human J_(H) non-coding sequences, each of which isadjacent to at least one of the one or more human J_(H) gene segments,wherein each of the one or more J_(H) non-coding sequences naturallyappears adjacent to a human J_(H) gene segment in an endogenous humanimmunoglobulin heavy chain locus; or

(iv) any combination thereof.

Embodiment 89

The isolated rodent cell of any one of embodiments 85-88, wherein theone or more rodent immunoglobulin heavy chain constant region genes areone or more endogenous rodent immunoglobulin heavy chain constant regiongenes.

Embodiment 90

The isolated rodent cell of any one of embodiments 85-89, wherein:

(i) the one or more human V_(H) gene segments comprise V_(H)3-74,V_(H)3-73, V_(H)3-72, V_(H)2-70, V_(H)1-69, V_(H)3-66, V_(H)3-64,V_(H)4-61, V_(H)4-59, V_(H)1-58, V_(H)3-53, V_(H)5-51, V_(H)3-49,V_(H)3-48, V_(H)1-46, V_(H)1-45, V_(H)3-43, V_(H)4-39, V_(H)4-34,V_(H)3-33, V_(H)4-31, V_(H)3-30, V_(H)4-28, V_(H)2-26, V_(H)1-24,V_(H)3-23, V_(H)3-21, V_(H)3-20, V_(H)1-18, V_(H)3-15, V_(H)3-13,V_(H)3-11, V_(H)3-9, V_(H)1-8, V_(H)3-7, V_(H)2-5, V_(H)7-4-1, V_(H)4-4,V_(H)1-3, V_(H)1-2, V_(H)6-1, or any combination thereof,

(ii) the one or more human D_(H) gene segments comprise D_(H)1-1,D_(H)2-2, D_(H)3-3, D_(H)4-4, D_(H)5-5, D_(H)6-6, D_(H)1-7, D_(H)2-8,D_(H)3-9, D_(H)3-10, D_(H)5-12, D_(H)6-13, D_(H)2-15, D_(H)3-16,D_(H)4-17, D_(H)6-19, D_(H)1-20, D_(H)2-21, D_(H)3-22, D_(H)6-25,D_(H)1-26, D_(H)7-27, or any combination thereof, and

(iii) the one or more human J_(H) gene segments comprise J_(H)1, J_(H)2,J_(H)3, J_(H)4, J_(H)5, J_(H)6, or any combination thereof.

Embodiment 91

The isolated rodent cell of any one of embodiments 85-90, wherein theengineered endogenous immunoglobulin heavy chain locus lacks afunctional endogenous rodent Adam6 gene.

Embodiment 92

The isolated rodent cell of any one of embodiments 85-91, wherein thegenome of the rodent cell further comprises one or more nucleotidesequences encoding one or more rodent ADAM6 polypeptides, functionalorthologs, functional homologs, or functional fragments thereof.

Embodiment 93

The isolated rodent cell of embodiment 92, wherein the one or morenucleotide sequences encoding one or more rodent ADAM6 polypeptides,functional orthologs, functional homologs, or functional fragmentsthereof are included on the same chromosome as the engineered endogenousimmunoglobulin heavy chain locus.

Embodiment 94

The isolated rodent cell of embodiment 92 or 93, wherein the one or morenucleotide sequences encoding one or more rodent ADAM6 polypeptides,functional orthologs, functional homologs, or functional fragmentsthereof are included in the engineered endogenous immunoglobulin heavychain locus.

Embodiment 95

The isolated rodent cell of any one of embodiments 92-94, wherein theone or more nucleotide sequences encoding one or more rodent ADAM6polypeptides, functional orthologs, functional homologs, or functionalfragments thereof are in place of a human Adam6 pseudogene.

Embodiment 96

The isolated rodent cell of any one of embodiments 92-95, wherein theone or more nucleotide sequences encoding one or more rodent ADAM6polypeptides, functional orthologs, functional homologs, or functionalfragments thereof replace a human Adam6 pseudogene.

Embodiment 97

The isolated rodent cell of any one of embodiments 92-96, wherein theone or more nucleotide sequences encoding one or more rodent ADAM6polypeptides, functional orthologs, functional homologs, or functionalfragments thereof are between a first human V_(H) gene segment and asecond human V_(H) gene segment.

Embodiment 98

The isolated rodent cell of embodiment 97, wherein the first human V_(H)gene segment is V_(H)1-2 and the second human V_(H) gene segment isV_(H)6-1.

Embodiment 99

The isolated rodent cell of any one of embodiments 92-94, wherein theone or more nucleotide sequences encoding one or more rodent ADAM6polypeptides, functional orthologs, functional homologs, or functionalfragments thereof are between a human V_(H) gene segment and a humanD_(H) gene segment.

Embodiment 100

The isolated rodent cell of any one embodiments 85-99, wherein theisolated rodent cell is homozygous for the engineered endogenousimmunoglobulin heavy chain locus.

Embodiment 101

The isolated rodent cell of any one of embodiments 71-100, wherein thegenome of the isolated rodent cell further comprises a nucleic acidsequence encoding an exogenous terminal deoxynucleotidyltransferase(TdT) operably linked to a transcriptional control element.

Embodiment 102

The isolated rodent cell of embodiment 101, wherein the TdT is a shortisoform of TdT (TdTS).

Embodiment 103

The isolated rodent cell of embodiment 101 or 102, wherein thetranscriptional control element comprises a RAG1 transcriptional controlelement, a RAG2 transcriptional control element, an immunoglobulin heavychain transcriptional control element, an immunoglobulin κ light chaintranscriptional control element, an immunoglobulin λ light chaintranscriptional control element, or any combination thereof.

Embodiment 104

The isolated rodent cell of any one of embodiments 101-103, wherein thenucleic acid sequence encoding an exogenous TdT is in the genome of therodent cell at an immunoglobulin κ light chain locus, an immunoglobulinλ light chain locus, an immunoglobulin heavy chain locus, a RAG1 locus,or a RAG2 locus.

Embodiment 105

The isolated rodent cell of any one of embodiments 71-104, wherein therodent cell is a rat cell or a mouse cell.

Embodiment 106

An immortalized cell generated from the isolated rodent cell of any oneof embodiments 71-105.

Embodiment 107

The isolated rodent cell of any one of embodiments 71-105, wherein therodent cell is a rodent embryonic stem (ES) cell.

Embodiment 108

A rodent embryo generated from the rodent ES cell of embodiment 107.

Embodiment 109

A method of making a genetically modified rodent comprising the stepsof:

(a) introducing one or more DNA fragments into the a first engineeredimmunoglobulin κ light chain locus in the genome of a rodent ES cell,wherein the one or more DNA fragments comprise:

-   -   (i) one or more human Vλ gene segments,    -   (ii) one or more human Jλ gene segments, and    -   (iii) a Cλ gene,    -   wherein the one or more human Vλ gene segments, the one or more        human Jλ gene segments, and the Cλ gene are introduced into the        genome of the rodent ES cell at the endogenous immunoglobulin κ        light chain locus, and wherein the one or more human Vλ gene        segments, the one or more human Jλ gene segments, and the Cλ        gene are operably linked; and

(b) generating a rodent using the rodent ES cell generated in (a).

Embodiment 110

The method of embodiment 109, wherein the genome of the rodent ES cellcomprises a second engineered endogenous immunoglobulin κ light chainlocus comprising:

(i) one or more human Vκ gene segments, and

(ii) one or more human Jκ gene segments,

wherein the one or more human Vκ gene segments and the one or more humanJκ gene segments are operably linked to a Cκ gene.

Embodiment 111

The method of embodiment 109 or 110, wherein the genome of the rodent EScell comprises an engineered endogenous immunoglobulin heavy chain locuscomprising:

(i) one or more human V_(H) gene segments,

(ii) one or more human D_(H) gene segments, and

(iii) one or more human J_(H) gene segments,

wherein the one or more human V_(H) gene segments, the one or more humanD_(H) gene segments, and the one or more human J_(H) gene segments areoperably linked to one or more rodent immunoglobulin heavy chainconstant region genes at the engineered endogenous immunoglobulin heavychain locus.

Embodiment 112

The method of any one of embodiments 109-111, wherein the one or moreDNA fragments further comprise at least one selection marker.

Embodiment 113

The method of any one of embodiments 109-112, wherein the one or moreDNA fragments further comprise at least one site-specific recombinationsite.

Embodiment 114

The method of any one of embodiments 109-113, further comprisingintroducing one or more human Vλ non-coding sequences and one or morehuman Vx non-coding sequences in the first engineered endogenousimmunoglobulin κ light chain locus, wherein each of the one or morehuman Vλ non-coding sequences is adjacent to a human Vx gene segment,and each of the one or more human Jλ non-coding sequences is adjacent toa human Jx gene segment.

Embodiment 115

The method of any one of embodiments 109-114, further comprisingintroducing a κ light chain non-coding sequence into the genome of therodent ES cell so that the K light chain non-coding sequence is betweenthe one or more human Vλ gene segments and the one or more human Jλ genesegments in the first engineered endogenous immunoglobulin κ light chainlocus.

Embodiment 116

The method of any one embodiments 109-115, wherein the rodent ES cell isa rat ES cell or a mouse ES cell.

Embodiment 117

A method of making a genetically modified rodent, comprising the stepof:

(a) engineering a first endogenous immunoglobulin κ light chain locus inthe germline genome of the rodent to include:

-   -   (i) one or more human Vλ gene segments,    -   (ii) one or more human Jλ gene segments, and    -   (iii) a Cλ gene,

wherein the one or more human Vλ gene segments and the one or more humanJλ gene segments are operably linked to the Cλ gene, and

wherein the Cλ gene is inserted at the first endogenous immunoglobulin κlight chain locus.

Embodiment 118

The method of embodiment 117, wherein the method further comprises thestep of:

(b) engineering a second endogenous immunoglobulin κ light chain locusin the germline genome of the rodent to include:

-   -   (i) one or more human Vλ gene segments,    -   (ii) one or more human Jλ gene segments, and    -   (iii) a Cλ gene,

wherein the one or more human Vλ gene segments and the one or more humanJλ gene segments are operably linked to the Cλ gene, and

wherein the Cλ gene is inserted at the second endogenous immunoglobulinκ light chain locus.

Embodiment 119

The method of embodiment 117, wherein the method further comprises thestep of:

(b) engineering a second engineered endogenous immunoglobulin κ lightchain locus in the germline genome of the rodent to include:

-   -   (i) one or more human Vκ gene segments, and    -   (ii) one or more human Jκ gene segments,

wherein the one or more human Vκ gene segments and the one or more humanJκ gene segments are operably linked to a Cκ gene.

Embodiment 120

The method of embodiment 119, wherein the Cκ gene at the secondengineered endogenous immunoglobulin κ light chain locus is anendogenous rodent Cκ gene.

Embodiment 121

The method of any one of embodiments 117-120, wherein the method furthercomprises the step of:

(c) engineering an engineered endogenous immunoglobulin heavy chainlocus in the germline genome of the rodent to include:

-   -   (i) one or more human V_(H) gene segments,    -   (ii) one or more human D_(H) gene segments, and    -   (iii) one or more human J_(H) gene segments,

wherein the one or more human V_(H) gene segments, the one or more humanD_(H) gene segments, and the one or more human J_(H) gene segments areoperably linked to one or more rodent immunoglobulin heavy chainconstant region genes.

Embodiment 122

The method of any one of embodiments 117-121, wherein the step ofengineering a first endogenous immunoglobulin κ light chain locus in thegermline genome of the rodent is carried out in a rodent ES cell whosegenome comprises a second engineered endogenous immunoglobulin κ lightchain locus comprising one or more human Vκ gene segments, and one ormore human Jκ gene segments, wherein the one or more human Vκ genesegments and the one or more human Jκ gene segments are operably linkedto a Cκ gene.

Embodiment 123

The method of embodiment 122, wherein the Cκ gene at the secondengineered endogenous immunoglobulin κ light chain locus is anendogenous rodent Cκ gene.

Embodiment 124

The method of any one of embodiments 117-120, 122 and 123, wherein thestep of engineering is carried out in a rodent ES cell whose genomecomprises an engineered endogenous immunoglobulin heavy chain locuscomprising one or more human V_(H) gene segments, one or more humanD_(H) gene segments, and one or more human J_(H) gene segments operablylinked to one or more rodent immunoglobulin heavy chain constant regiongenes.

Embodiment 125

The method of any one of embodiments 121-124, wherein the engineeredendogenous immunoglobulin heavy chain locus comprises one or more humanV_(H) non-coding sequences, each of which is adjacent to at least one ofthe one or more human V_(H) gene segments, wherein each of the one ormore human V_(H) non-coding sequences naturally appears adjacent to ahuman V_(H) gene segment in an endogenous human immunoglobulin heavychain locus.

Embodiment 126

The method of any one of embodiments 121-125, wherein the engineeredendogenous immunoglobulin heavy chain locus comprises one or more humanD_(H) non-coding sequences, each of which is adjacent to at least one ofthe one or more human D_(H) gene segments, wherein each of the one ormore D_(H) non-coding sequences naturally appears adjacent to a humanD_(H) gene segment in an endogenous human immunoglobulin heavy chainlocus.

Embodiment 127

The method of any one of embodiments 121-126, wherein the engineeredendogenous immunoglobulin heavy chain locus comprises one or more humanJ_(H) non-coding sequences, each of which is adjacent to at least one ofthe one or more human J_(H) gene segments, wherein each of the one ormore J_(H) non-coding sequences naturally appears adjacent to a humanJ_(H) gene segment in an endogenous human immunoglobulin heavy chainlocus.

Embodiment 128

The method of any one of embodiments 109-127, wherein the firstengineered endogenous immunoglobulin κ light chain locus lacks a rodentCκ gene.

Embodiment 129

The method of any one of embodiments 110-115 andll119-128, wherein theCκ gene at the second engineered endogenous immunoglobulin κ light chainlocus is an endogenous rodent Cκ gene.

Embodiment 130

The method of any one of embodiments 109-129, wherein the Cλ gene at thefirst engineered endogenous immunoglobulin κ light chain locus comprisesa rodent Cλ gene.

Embodiment 131

The method of any one of embodiments 109-130, wherein the firstengineered endogenous immunoglobulin κ light chain locus furthercomprises:

(i) one or more human Vλ non-coding sequences, each of which is adjacentto at least one of the one or more human Vλ gene segments, wherein theone or more human Vλ non-coding sequences naturally appears adjacent toa human Vλ gene segment in an endogenous human immunoglobulin λ lightchain locus;

(ii) one or more human Jλ non-coding sequences, each of which isadjacent to at least one of the one or more human Jλ gene segments,wherein the one or more human Jλ non-coding sequences naturally appearsadjacent to a human Jλ gene segment in an endogenous humanimmunoglobulin λ light chain locus; or

(iii) any combination thereof.

Embodiment 132

The method of any one of embodiments 109-131, wherein:

(i) the one or more human Vλ gene segments comprise Vλ5-52, Vλ1-51,Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37,Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14,Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, Vλ3-1, or any combinationthereof; and

(ii) the one or more human Jλ gene segments comprise Jλ1, Jλ2, Jλ3, Jλ6,Jλ7, or any combination thereof.

Embodiment 133

The method of embodiment 132, wherein the first engineered endogenousimmunoglobulin κ light chain locus comprises:

(i) one or more human Vλ non-coding sequences, wherein each of the oneor more human Vλ non-coding sequences is adjacent to the Vλ5-52, Vλ1-51,Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37,Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14,Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, or Vλ3-1 in the firstengineered endogenous immunoglobulin κ light chain locus, and whereineach of the one or more human Vλ non-coding sequences naturally appearsadjacent to a Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44,Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22,Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8,Vλ4-3, or Vλ3-1 of an endogenous human immunoglobulin λ light chainlocus; and

(ii) one or more human Jλ non-coding sequences, wherein each of the oneor more human Jλ non-coding sequences is adjacent to the Jλ1, Jλ2, Jλ3,Jλ6 or Jλ7 in the first engineered endogenous immunoglobulin κ lightchain locus, and wherein each of the one or more human Jλ non-codingsequences naturally appears adjacent to a Jλ1, Jλ2, Jλ3, Jλ6 or Jλ7 ofan endogenous human immunoglobulin λ light chain locus.

Embodiment 134

The method of embodiment 132, wherein the first engineered endogenousimmunoglobulin κ light chain locus comprises:

(i) one or more human Vλ non-coding sequences, wherein each of the oneor more human Vλ non-coding sequences is adjacent to the Vλ5-52, Vλ1-51,Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37,Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16,Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3 or Vλ3-1 in thefirst engineered endogenous immunoglobulin κ light chain locus, andwherein each of the one or more human Vλ non-coding sequences naturallyappears adjacent to a Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45,Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23,Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10,Vλ3-9, Vλ2-8, Vλ4-3 or Vλ3-1 of an endogenous human immunoglobulin λlight chain locus; and

(ii) one or more human Jκ non-coding sequences, wherein each of the oneor more human Jκ non-coding sequences is adjacent to the Jλ1, Jλ2, Jλ3,Jλ6 or Jλ7 in the first engineered endogenous immunoglobulin κ lightchain locus, and wherein each of the one or more human Jκ non-codingsequences naturally appears adjacent to a Jκ1, Jκ2, Jκ3, Jκ4, or Jκ5 ofan endogenous human immunoglobulin κ light chain locus.

Embodiment 135

The method of any one of embodiments 109-134, wherein:

(i) the one or more human Vλ gene segments comprise Vλ4-69, Vλ8-61,Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45,Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23,Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9,Vλ2-8, Vλ4-3, Vλ3-1, or any combination thereof; and

(ii) the one or more human Jλ gene segments comprise Jλ1, Jλ2, Jλ3, Jλ6,Jλ7, or any combination thereof.

Embodiment 136

The method of embodiment 135, wherein the first engineered endogenousimmunoglobulin κ light chain locus comprises:

(i) one or more human Vλ non-coding sequences, wherein each of the oneor more human Vλ non-coding sequences is adjacent to the Vλ4-69, Vλ8-61,Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45,Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23,Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9,Vλ2-8, Vλ4-3, or Vλ3-1 in the first engineered endogenous immunoglobulinκ light chain locus, and wherein each of the one or more human Vλnon-coding sequences naturally appears adjacent to a Vλ4-69, Vλ8-61,Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45,Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23,Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9,Vλ2-8, Vλ4-3, or Vλ3-1 of an endogenous human immunoglobulin λ lightchain locus; and

(ii) one or more human Jλ non-coding sequences, wherein each of the oneor more human Jλ non-coding sequences is adjacent to the Jλ1, Jλ2, Jλ3,Jλ6 or Jλ7 in the first engineered endogenous immunoglobulin κ lightchain locus, and wherein each of the one or more human Jλ non-codingsequences naturally appears adjacent to a Jλ1, Jλ2, Jλ3, Jλ6 or Jλ7 ofan endogenous human immunoglobulin λ light chain locus.

Embodiment 137

The method of embodiment 135, wherein the first engineered endogenousimmunoglobulin κ light chain locus comprises:

(i) one or more human Vλ non-coding sequences, wherein each of the oneor more human Vλ non-coding sequences is adjacent to the Vλ4-69, Vλ8-61,Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45,Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23,Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10,Vλ3-9, Vλ2-8, Vλ4-3 or Vλ3-1 in the first engineered endogenousimmunoglobulin κ light chain locus, and wherein each of the one or morehuman Vλ non-coding sequences naturally appears adjacent to a Vλ4-69,Vλ8-61, Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46,Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25,Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11,Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3 or Vλ3-1 of an endogenous humanimmunoglobulin λ light chain locus; and

(ii) one or more human Jκ non-coding sequences, wherein each of the oneor more human Jκ non-coding sequences is adjacent to the Jλ1, Jλ2, Jλ3,Jλ6 or Jλ7 in the first engineered endogenous immunoglobulin κ lightchain locus, and wherein each of the one or more human Jκ non-codingsequences naturally appears adjacent to a Jκ1, Jκ2, Jκ3, Jκ4, or Jκ5 ofan endogenous human immunoglobulin κ light chain locus.

Embodiment 138

The method of any one of embodiments 111-116 and 121-137, wherein theone or more human V_(H) gene segments, one or more human D_(H) genesegments, and one or more human J_(H) gene segments are in place of oneor more rodent V_(H) gene segments, one or more rodent D_(H) genesegments, one or more rodent J_(H) gene segments, or a combinationthereof.

Embodiment 139

The method of any one of embodiments 111-116 and 121-138, wherein theone or more human V_(H) gene segments, one or more human D_(H) genesegments, and one or more human J_(H) gene segments replace one or morerodent V_(H) gene segments, one or more rodent D_(H) gene segments, oneor more rodent J_(H) gene segments, or any combination thereof.

Embodiment 140

The method of any one of embodiments 111-116 and 121-139, wherein theengineered endogenous immunoglobulin heavy chain locus furthercomprises:

(i) one or more human V_(H) non-coding sequences, each of which isadjacent to at least one of the one or more human V_(H) gene segments,wherein each of the one or more V_(H) non-coding sequences naturallyappears adjacent to a human V_(H) gene segment in an endogenous humanimmunoglobulin heavy chain locus;

(ii) one or more human D_(H) non-coding sequences, each of which isadjacent to at least one of the one or more human D_(H) gene segments,wherein each of the one or more D_(H) non-coding sequences naturallyappears adjacent to a human D_(H) gene segment in an endogenous humanimmunoglobulin heavy chain locus;

(iii) one or more human J_(H) non-coding sequences, each of which isadjacent to at least one of the one or more human J_(H) gene segments,wherein each of the one or more J_(H) non-coding sequences naturallyappears adjacent to a human J_(H) gene segment in an endogenous humanimmunoglobulin heavy chain locus; or

(iv) any combination thereof.

Embodiment 141

The method of any one of embodiments 111-116 and 121-140, wherein theone or more rodent immunoglobulin heavy chain constant region genes areone or more endogenous rodent immunoglobulin heavy chain constant regiongenes.

Embodiment 142

The method of any one of embodiments 111-116 and 121-141, wherein:

(i) the one or more human V_(H) gene segments comprise V_(H)3-7⁴,V_(H)3-7³, V_(H)3-7², V_(H)2-70, V_(H)1-69, V_(H)3-66, V_(H)3-64,V_(H)4-61, V_(H)4-59, V_(H)1-58, V_(H)3-53, V_(H)5-51, V_(H)3-49,V_(H)3-48, V_(H)1-46, V_(H)1-45, V_(H)3-43, V_(H)4-39, V_(H)4-34,V_(H)3-33, V_(H)4-31, V_(H)3-30, V_(H)4-28, V_(H)2-26, V_(H)1-24,V_(H)3-23, V_(H)3-21, V_(H)3-20, V_(H)1-18, V_(H)3-15, V_(H)3-13,V_(H)3-11, V_(H)3-9, V_(H)1-8, V_(H)3-7, V_(H)2-5, V_(H)7-4-1, V_(H)4-4,V_(H)1-3, V_(H)1-2, V_(H)6-1, or any combination thereof;

(ii) the one or more human D_(H) gene segments comprise D_(H)1-1,D_(H)2-2, D_(H)3-3, D_(H)4-4, D_(H)5-5, D_(H)6-6, D_(H)1-7, D_(H)2-8,D_(H)3-9, D_(H)3-10, D_(H)5-12, D_(H)6-13, D_(H)2-15, D_(H)3-16,D_(H)4-17, D_(H)6-19, D_(H)1-20, D_(H)2-21, D_(H)3-22, D_(H)6-25,D_(H)1-26, D_(H)7-27, or any combination thereof; and

(iii) the one or more human J_(H) gene segments comprise J_(H)1, J_(H)2,J_(H)3, J_(H)4, J_(H)5, J_(H)6, or any combination thereof.

Embodiment 143

The method of any one of embodiments 111-116 and 121-142, wherein theengineered endogenous immunoglobulin heavy chain locus lacks afunctional endogenous rodent Adam6 gene.

Embodiment 144

The method of any one of embodiments 111-116 and 121-143, wherein thegenome of the rodent ES cell or the germline genome of the rodentfurther comprises one or more nucleotide sequences encoding one or morerodent ADAM6 polypeptides, functional orthologs, functional homologs, orfunctional fragments thereof.

Embodiment 145

The method of embodiment 144, wherein the one or more nucleotidesequences encoding one or more rodent ADAM6 polypeptides, functionalorthologs, functional homologs, or functional fragments thereof areincluded on the same chromosome as the engineered endogenousimmunoglobulin heavy chain locus.

Embodiment 146

The method of embodiment 144 or 145, wherein the one or more nucleotidesequences encoding one or more rodent ADAM6 polypeptides, functionalorthologs, functional homologs, or functional fragments thereof areincluded in the engineered endogenous immunoglobulin heavy chain locus.

Embodiment 147

The method of any one of embodiments 144-146, wherein the one or morenucleotide sequences encoding one or more rodent ADAM6 polypeptides,functional orthologs, functional homologs, or functional fragmentsthereof are in place of a human Adam6 pseudogene.

Embodiment 148

The method of any one of embodiments 144-147, wherein the one or morenucleotide sequences encoding one or more rodent ADAM6 polypeptides,functional orthologs, functional homologs, or functional fragmentsthereof replace a human Adam6 pseudogene.

Embodiment 149

The method of any one of embodiments 144-148, wherein the one or morenucleotide sequences encoding one or more rodent ADAM6 polypeptides,functional orthologs, functional homologs, or functional fragmentsthereof are between a first human V_(H) gene segment and a second humanV_(H) gene segment.

Embodiment 150

The method of embodiment 149, wherein the first human V_(H) gene segmentis V_(H)1-2 and the second human V_(H) gene segment is V_(H)6-1.

Embodiment 151

The method of any one of embodiments 144-146, wherein the one or morenucleotide sequences encoding one or more rodent ADAM6 polypeptides,functional orthologs, functional homologs, or functional fragmentsthereof are between a human V_(H) gene segment and a human D_(H) genesegment.

Embodiment 152

The method of any one embodiments 111-116 and 121-151, wherein therodent is homozygous for the engineered endogenous immunoglobulin heavychain locus.

Embodiment 153

The method of any one of embodiments 109-152, wherein the genome of therodent ES cell or the germline genome of the rodent further comprises anucleic acid sequence encoding an exogenous terminaldeoxynucleotidyltransferase (TdT) operably linked to a transcriptionalcontrol element.

Embodiment 154

The method of embodiment 153, wherein the TdT is a short isoform of TdT(TdTS).

Embodiment 155

The method of embodiment 153 or 154, wherein the transcriptional controlelement comprises a RAG1 transcriptional control element, a RAG2transcriptional control element, an immunoglobulin heavy chaintranscriptional control element, an immunoglobulin κ light chaintranscriptional control element, an immunoglobulin λ light chaintranscriptional control element, or any combination thereof.

Embodiment 156

The method of any one of embodiments 153-155, wherein the nucleic acidsequence encoding an exogenous TdT is at an immunoglobulin κ light chainlocus, an immunoglobulin λ light chain locus, an immunoglobulin heavychain locus, a RAG1 locus, or a RAG2 locus.

Embodiment 157

The method of any one embodiments 109-156, wherein the rodent is a rator a mouse.

Embodiment 158

A method of producing an antibody in a genetically modified rodent, themethod comprising the steps of:

(a) immunizing a rodent with an antigen of interest,

wherein the rodent has a germline genome comprising:

-   -   a first engineered endogenous immunoglobulin κ light chain        locus, comprising:        -   (i) one or more human Vλ gene segments,        -   (ii) one or more human Jλ gene segments, and        -   (iii) a Cλ gene,    -   wherein the one or more human Vλ gene segments and the one or        more human Jλ gene segments are operably linked to the Cλ gene,        and    -   wherein the Cλ gene (c) is in the place of a rodent Cκ gene at        the first engineered endogenous immunoglobulin κ light chain        locus;

(b) maintaining the rodent under conditions sufficient for the rodent toproduce an immune response to the antigen of interest; and

(c) recovering from the rodent:

-   -   (i) an antibody that binds the antigen of interest,    -   (ii) a nucleotide that encodes a human light or heavy chain        variable domain, a light chain, or a heavy chain of an antibody        that binds the antigen of interest, or    -   (iii) a cell that expresses an antibody that binds the antigen        of interest.

Embodiment 159

The method of embodiment 158, wherein the cell of the rodent is a Bcell.

Embodiment 160

The method of embodiment 159, further comprising producing a hybridomafrom the B cell.

Embodiment 161

A method of making an antibody, comprising:

(a) expressing a first nucleotide sequence that encodes animmunoglobulin heavy chain in a host cell, wherein the first nucleotidesequence includes a human heavy chain variable region sequence;

(b) expressing a second nucleotide sequence that encodes animmunoglobulin λ light chain in a host cell, wherein the secondnucleotide sequence includes a human λ light chain variable regionsequence that was identified from a genetically modified rodent whosegermline genome comprises:

-   -   a first engineered endogenous immunoglobulin κ light chain locus        comprising:        -   (i) one or more human Vλ gene segment,        -   (ii) one or more human Jλ gene segment, and        -   (iii) a Cλ gene,    -   wherein the one or more human Vλ gene segment and the one or        more human Jλ gene segment are operably linked to the Cλ gene,        and    -   wherein the rodent lacks a rodent Cκ gene at the engineered        endogenous immunoglobulin κ light chain locus;

(c) culturing the host cell so that immunoglobulin light chains andimmunoglobulin heavy chains are expressed and form an antibody; and

(d) obtaining the antibody from the host cell or host cell culture.

Embodiment 162

The method of embodiment 161, wherein the first nucleotide furtherincludes a human heavy chain constant region gene.

Embodiment 163

The method of embodiment 161 or 162, wherein the second nucleotidefurther includes a human λ light chain constant region gene.

Embodiment 164

The method of any one of embodiments 158-163, wherein the rodent ishomozygous for the first engineered endogenous immunoglobulin κ lightchain locus.

Embodiment 165

The method of any one of embodiments 158-163, wherein the rodent isheterozygous for the first engineered endogenous immunoglobulin κ lightchain locus.

Embodiment 166

The method of embodiment 165, wherein the germline genome of the rodentcomprises a second engineered endogenous immunoglobulin κ light chainlocus comprising:

(a) one or more human Vκ gene segments, and

(b) one or more human Jκ gene segments,

wherein the one or more human Vκ gene segments and the one or more humanJκ gene segments are operably linked to a Cκ gene.

Embodiment 167

The method of embodiment 166, wherein the Cκ gene at the secondengineered endogenous immunoglobulin κ light chain locus is anendogenous rodent Cκ gene.

Embodiment 168

The method of any one of embodiments 158-167, wherein the rodent lacks arodent Cκ gene at the first engineered endogenous immunoglobulin κ lightchain locus.

Embodiment 169

The method of any one of embodiments 158-168, wherein the Cλ gene at thefirst engineered endogenous immunoglobulin κ light chain locus comprisesa rodent Cλ gene.

Embodiment 170

The method of any one of embodiments 158-169, wherein the firstengineered endogenous immunoglobulin κ light chain locus furthercomprises:

(i) one or more human Vλ non-coding sequences, each of which is adjacentto at least one of the one or more human Vλ gene segments, wherein theone or more human Vλ non-coding sequences naturally appears adjacent toa human Vλ gene segment in an endogenous human immunoglobulin λ lightchain locus;

(ii) one or more human Jλ non-coding sequences, each of which isadjacent to at least one of the one or more human Jλ gene segments,wherein the one or more human Jλ non-coding sequences naturally appearsadjacent to a human Jλ gene segment in an endogenous humanimmunoglobulin λ light chain locus; or

(iii) any combination thereof.

Embodiment 171

The method of any one of embodiments 158-170, wherein:

(i) the one or more human Vλ gene segments comprise Vλ5-52, Vλ1-51,Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37,Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14,Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, Vλ3-1, or any combinationthereof; and

(ii) the one or more human Jλ gene segments comprise Jλ1, Jλ2, Jλ3, Jλ6,Jλ7, or any combination thereof.

Embodiment 172

The method of embodiment 171, wherein the first engineered endogenousimmunoglobulin κ light chain locus comprises:

(i) one or more human Vλ non-coding sequences, wherein each of the oneor more human Vλ non-coding sequences is adjacent to the Vλ5-52, Vλ1-51,Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37,Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14,Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, or Vλ3-1 in the firstengineered endogenous immunoglobulin κ light chain locus, and whereineach of the one or more human Vλ non-coding sequences naturally appearsadjacent to a Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44,Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22,Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8,Vλ4-3, or Vλ3-1 of an endogenous human immunoglobulin λ light chainlocus; and

(ii) one or more human Jλ non-coding sequences, wherein each of the oneor more human Jλ non-coding sequences is adjacent to the Jλ1, Jλ2, Jλ3,Jλ6 or Jλ7 in the first engineered endogenous immunoglobulin κ lightchain locus, and wherein each of the one or more human Jx non-codingsequences naturally appears adjacent to a Jλ1, Jλ2, Jλ3, Jλ6 or Jλ7 ofan endogenous human immunoglobulin λ light chain locus.

Embodiment 173

The method of embodiment 171, wherein the first engineered endogenousimmunoglobulin κ light chain locus comprises:

(i) one or more human Vλ non-coding sequences, wherein each of the oneor more human Vλ non-coding sequences is adjacent to the Vλ5-52, Vλ1-51,Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37,Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16,Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3 or Vλ3-1 in thefirst engineered endogenous immunoglobulin κ light chain locus, andwherein each of the one or more human Vλ non-coding sequences naturallyappears adjacent to a Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45,Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23,Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10,Vλ3-9, Vλ2-8, Vλ4-3 or Vλ3-1 of an endogenous human immunoglobulin λlight chain locus; and

(ii) one or more human Jκ non-coding sequences, wherein each of the oneor more human Jκ non-coding sequences is adjacent to the Jλ1, Jλ2, Jλ3,Jλ6 or Jλ7 in the first engineered endogenous immunoglobulin κ lightchain locus, and wherein each of the one or more human Jκ non-codingsequences naturally appears adjacent to a Jκ1, Jκ2, Jκ3, Jκ4, or Jκ5 ofan endogenous human immunoglobulin κ light chain locus.

Embodiment 174

The method of any one of embodiments 158-173, wherein:

(i) the one or more human Vλ gene segments comprise Vλ4-69, Vλ8-61,Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45,Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23,Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9,Vλ2-8, Vλ4-3, Vλ3-1, or any combination thereof; and

(ii) the one or more human Jλ gene segments comprise Jλ1, Jλ2, Jλ3, Jλ6,Jλ7, or any combination thereof.

Embodiment 175

The method of embodiment 174, wherein the first engineered endogenousimmunoglobulin κ light chain locus comprises:

(i) one or more human Vλ non-coding sequences, wherein each of the oneor more human Vλ non-coding sequences is adjacent to the Vλ4-69, Vλ8-61,Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45,Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23,Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9,Vλ2-8, Vλ4-3, or Vλ3-1 in the first engineered endogenous immunoglobulinκ light chain locus, and wherein each of the one or more human Vλnon-coding sequences naturally appears adjacent to a Vλ4-69, Vλ8-61,Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45,Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23,Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9,Vλ2-8, Vλ4-3, or Vλ3-1 of an endogenous human immunoglobulin λ lightchain locus; and

(ii) one or more human Jλ non-coding sequences, wherein each of the oneor more human Jλ non-coding sequences is adjacent to the Jλ1, Jλ2, Jλ3,Jλ6 or Jλ7 in the first engineered endogenous immunoglobulin κ lightchain locus, and wherein each of the one or more human Jλ non-codingsequences naturally appears adjacent to a Jλ1, Jλ2, Jλ3, Jλ6 or Jλ7 ofan endogenous human immunoglobulin λ light chain locus.

Embodiment 176

The method of embodiment 174, wherein the first engineered endogenousimmunoglobulin κ light chain locus comprises:

(i) one or more human Vλ non-coding sequences, wherein each of the oneor more human Vλ non-coding sequences is adjacent to the Vλ4-69, Vλ8-61,Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45,Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23,Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10,Vλ3-9, Vλ2-8, Vλ4-3 or Vλ3-1 in the first engineered endogenousimmunoglobulin κ light chain locus, and wherein each of the one or morehuman Vλ non-coding sequences naturally appears adjacent to a Vλ4-69,Vλ8-61, Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46,Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25,Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11,Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3 or Vλ3-1 of an endogenous humanimmunoglobulin λ light chain locus,

(ii) one or more human Jκ non-coding sequences, wherein each of the oneor more human Jκ non-coding sequences is adjacent to the Jλ1, Jλ2, Jλ3,Jλ6 or Jλ7 in the first engineered endogenous immunoglobulin κ lightchain locus, and wherein each of the one or more human Jκ non-codingsequences naturally appears adjacent to a Jκ1, Jκ2, Jκ3, Jκ4, or Jκ5 ofan endogenous human immunoglobulin κ light chain locus.

Embodiment 177

The method of any one of embodiments 158-176, wherein the germlinegenome of the rodent further comprises:

an engineered endogenous immunoglobulin heavy chain locus, comprising:

-   -   (a) one or more human V_(H) gene segments,    -   (b) one or more human D_(H) gene segments, and    -   (c) one or more human J_(H) gene segments,

wherein the one or more human V_(H) gene segments, the one or more humanD_(H) gene segments, and the one or more human J_(H) gene segments areoperably linked to one or more rodent immunoglobulin heavy chainconstant region genes at the engineered endogenous immunoglobulin heavychain locus.

Embodiment 178

The method of embodiment 177, wherein the one or more human V_(H) genesegments, one or more human D_(H) gene segments, and one or more humanJ_(H) gene segments are in place of one or more rodent V_(H) genesegments, one or more rodent D_(H) gene segments, one or more rodentJ_(H) gene segments, or a combination thereof.

Embodiment 179

The method of embodiment 177 or 178, wherein the one or more human V_(H)gene segments, one or more human D_(H) gene segments, and one or morehuman J_(H) gene segments replace one or more rodent V_(H) genesegments, one or more rodent D_(H) gene segments, one or more rodentJ_(H) gene segments, or any combination thereof.

Embodiment 180

The method of any one of embodiments 177-179, wherein the engineeredendogenous immunoglobulin heavy chain locus further comprises:

(i) one or more human V_(H) non-coding sequences, each of which isadjacent to at least one of the one or more human V_(H) gene segments,wherein each of the one or more V_(H) non-coding sequences naturallyappears adjacent to a human V_(H) gene segment in an endogenous humanimmunoglobulin heavy chain locus;

(ii) one or more human D_(H) non-coding sequences, each of which isadjacent to at least one of the one or more human D_(H) gene segments,wherein each of the one or more D_(H) non-coding sequences naturallyappears adjacent to a human D_(H) gene segment in an endogenous humanimmunoglobulin heavy chain locus;

(iii) one or more human J_(H) non-coding sequences, each of which isadjacent to at least one of the one or more human J_(H) gene segments,wherein each of the one or more J_(H) non-coding sequences naturallyappears adjacent to a human J_(H) gene segment in an endogenous humanimmunoglobulin heavy chain locus; or

(iv) any combination thereof.

Embodiment 181

The method of any one of embodiments 177-180, wherein the one or morerodent immunoglobulin heavy chain constant region genes are one or moreendogenous rodent immunoglobulin heavy chain constant region genes.

Embodiment 182

The method of any one of embodiments 177-181, wherein:

(i) the one or more human V_(H) gene segments comprise V_(H)3-7⁴,V_(H)3-7³, V_(H)3-7², V_(H)2-70, V_(H)1-69, V_(H)3-66, V_(H)3-64,V_(H)4-61, V_(H)4-59, V_(H)1-58, V_(H)3-53, V_(H)5-51, V_(H)3-49,V_(H)3-48, V_(H)1-46, V_(H)1-45, V_(H)3-43, V_(H)4-39, V_(H)4-34,V_(H)3-33, V_(H)4-31, V_(H)3-30, V_(H)4-28, V_(H)2-26, V_(H)1-24,V_(H)3-23, V_(H)3-21, V_(H)3-20, V_(H)1-18, V_(H)3-15, V_(H)3-13,V_(H)3-11, V_(H)3-9, V_(H)1-8, V_(H)3-7, V_(H)2-5, V_(H)7-4-1, V_(H)4-4,V_(H)1-3, V_(H)1-2, V_(H)6-1, or any combination thereof,

(ii) the one or more human D_(H) gene segments comprise D_(H)1-1,D_(H)2-2, D_(H)3-3, D_(H)4-4, D_(H)5-5, D_(H)6-6, D_(H)1-7, D_(H)2-8,D_(H)3-9, D_(H)3-10, D_(H)5-12, D_(H)6-13, D_(H)2-15, D_(H)3-16,D_(H)4-17, D_(H)6-19, D_(H)1-20, D_(H)2-21, D_(H)3-22, D_(H)6-25,D_(H)1-26, D_(H)7-27, or any combination thereof, and

(iii) the one or more human J_(H) gene segments comprise J_(H)1, J_(H)2,J_(H)3, J_(H)4, J_(H)5, J_(H)6, or any combination thereof.

Embodiment 183

The method of any one of embodiments 177-182, wherein the engineeredendogenous immunoglobulin heavy chain locus lacks a functionalendogenous rodent Adam6 gene.

Embodiment 184

The method of any one of embodiments 177-183, wherein the germlinegenome of the rodent further comprises one or more nucleotide sequencesencoding one or more rodent ADAM6 polypeptides, functional orthologs,functional homologs, or functional fragments thereof.

Embodiment 185

The method of embodiment 184, wherein the one or more rodent ADAM6polypeptides, functional orthologs, functional homologs, or functionalfragments thereof are expressed.

Embodiment 186

The method of embodiment 184 or 185, wherein the one or more nucleotidesequences encoding one or more rodent ADAM6 polypeptides, functionalorthologs, functional homologs, or functional fragments thereof areincluded on the same chromosome as the engineered endogenousimmunoglobulin heavy chain locus.

Embodiment 187

The method of any one of embodiments 184-186, wherein the one or morenucleotide sequences encoding one or more rodent ADAM6 polypeptides,functional orthologs, functional homologs, or functional fragmentsthereof are included in the engineered endogenous immunoglobulin heavychain locus.

Embodiment 188

The method of any one of embodiments 184-187, wherein the one or morenucleotide sequences encoding one or more rodent ADAM6 polypeptides,functional orthologs, functional homologs, or functional fragmentsthereof are in place of a human Adam6 pseudogene.

Embodiment 189

The method of any one of embodiments 184-188, wherein the one or morenucleotide sequences encoding one or more rodent ADAM6 polypeptides,functional orthologs, functional homologs, or functional fragmentsthereof replace a human Adam6 pseudogene.

Embodiment 190

The method of any one of embodiments 184-189, wherein the one or morenucleotide sequences encoding one or more rodent ADAM6 polypeptides,functional orthologs, functional homologs, or functional fragmentsthereof are between a first human V_(H) gene segment and a second humanV_(H) gene segment.

Embodiment 191

The method of embodiment 190, wherein the first human V_(H) gene segmentis V_(H)1-2 and the second human V_(H) gene segment is V_(H)6-1.

Embodiment 192

The method of any one of embodiments 184-187, wherein the one or morenucleotide sequences encoding one or more rodent ADAM6 polypeptides,functional orthologs, functional homologs, or functional fragmentsthereof are between a human V_(H) gene segment and a human D_(H) genesegment.

Embodiment 193

The method of any one embodiments 177-192, wherein the rodent ishomozygous for the engineered endogenous immunoglobulin heavy chainlocus.

Embodiment 194

The method of any one of embodiments 158-193, wherein the rodentcomprises a population of B cells that express antibodies, includingimmunoglobulin λ light chains that each include a human immunoglobulin λlight chain variable domain.

Embodiment 195

The method of embodiment 194, wherein the human immunoglobulin λ lightchain variable domain is encoded by a rearranged human immunoglobulin λlight chain variable region sequence comprising (i) one of the one ormore human Vλ gene segments or a somatically hypermutated variantthereof, and (ii) one of the one or more human Jλ gene segments or asomatically hypermutated variant thereof.

Embodiment 196

The method of any one of embodiments 158-195, wherein the germlinegenome of the rodent further comprises a nucleic acid sequence encodingan exogenous terminal deoxynucleotidyltransferase (TdT) operably linkedto a transcriptional control element.

Embodiment 197

The method of embodiment 196, wherein the TdT is a short isoform of TdT(TdTS).

Embodiment 198

The method of embodiment 196 or 197, wherein the transcriptional controlelement comprises a RAG1 transcriptional control element, a RAG2transcriptional control element, an immunoglobulin heavy chaintranscriptional control element, an immunoglobulin κ light chaintranscriptional control element, an immunoglobulin λ light chaintranscriptional control element, or any combination thereof.

Embodiment 199

The method of any one of embodiments 196-198, wherein the nucleic acidsequence encoding an exogenous TdT is in the germline genome at animmunoglobulin κ light chain locus, an immunoglobulin λ light chainlocus, an immunoglobulin heavy chain locus, a RAG1 locus, or a RAG2locus.

Embodiment 200

The method any one of embodiments 158-199, wherein the rodent is a rator a mouse.

Embodiment 201

A rodent embryonic stem (ES) cell, whose genome comprises:

-   -   a first engineered endogenous immunoglobulin κ light chain locus        comprising:        -   (a) one or more human Vλ gene segments,        -   (b) one or more human Jλ gene segments, and        -   (c) a Cλ gene,    -   wherein the one or more human Vλ gene segments and the one or        more human Jλ gene segments are operably linked to the Cλ gene,        and

wherein the rodent ES cell lacks a rodent Cκ gene at the firstengineered endogenous immunoglobulin κ light chain locus.

Embodiment 202

The rodent ES cell of embodiment 201, wherein the rodent ES cell ishomozygous for the first engineered endogenous immunoglobulin κ lightchain locus.

Embodiment 203

The rodent ES cell of embodiment 201, wherein the rodent ES cell isheterozygous for the first engineered endogenous immunoglobulin κ lightchain locus.

Embodiment 204

The rodent ES cell of embodiment 203, wherein the genome of the rodentES cell comprises a second engineered endogenous immunoglobulin κ lightchain locus comprising:

(i) one or more human Vκ gene segments, and

(ii) one or more human Jκ gene segments,

wherein the one or more human Vκ gene segments and the one or more humanJκ gene segments are operably linked to a Cκ gene.

Embodiment 205

The rodent ES cell of embodiment 204, wherein the Cκ gene at the secondengineered endogenous immunoglobulin κ light chain locus is anendogenous rodent Cκ gene.

Embodiment 206

The rodent ES cell of any one of embodiments 201-205, wherein the genomeof the rodent ES cell comprises an engineered endogenous immunoglobulinheavy chain locus comprising:

(i) one or more human V_(H) gene segments,

(ii) one or more human D_(H) gene segments, and

(iii) one or more human J_(H) gene segments,

wherein the one or more human V_(H) gene segments, the one or more humanD_(H) gene segments, and the one or more human J_(H) gene segments areoperably linked to one or more rodent immunoglobulin heavy chainconstant region genes at the engineered endogenous immunoglobulin heavychain locus.

Embodiment 207

The rodent ES cell of embodiments 201-206, wherein the rodent lacks arodent Cκ gene at the first engineered endogenous immunoglobulin κ lightchain locus.

Embodiment 208

The rodent ES cell of any one of embodiments 201-207, wherein the Cλgene at the first engineered endogenous immunoglobulin κ light chainlocus comprises a rodent Cλ gene.

Embodiment 209

The rodent ES cell of any one of embodiments 201-208, wherein the firstengineered endogenous immunoglobulin κ light chain locus furthercomprises:

(i) one or more human Vλ non-coding sequences, each of which is adjacentto at least one of the one or more human Vλ gene segments, wherein theone or more human Vλ non-coding sequences naturally appears adjacent toa human Vλ gene segment in an endogenous human immunoglobulin λ lightchain locus;

(ii) one or more human Jλ non-coding sequences, each of which isadjacent to at least one of the one or more human Jλ gene segments,wherein the one or more human Jλ non-coding sequences naturally appearsadjacent to a human Jλ gene segment in an endogenous humanimmunoglobulin λ light chain locus; or

(iii) any combination thereof.

Embodiment 210

The rodent ES cell of any one of embodiments 201-209, wherein:

(i) the one or more human Vλ gene segments comprise Vλ5-52, Vλ1-51,Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37,Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14,Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, Vλ3-1, or any combinationthereof; and

(ii) the one or more human Jλ gene segments comprise Jλ1, Jλ2, Jλ3, Jλ6,Jλ7, or any combination thereof.

Embodiment 211

The rodent ES cell of embodiment 210, wherein the first engineeredendogenous immunoglobulin κ light chain locus comprises:

(i) one or more human Vλ non-coding sequences, wherein each of the oneor more human Vλ non-coding sequences is adjacent to the Vλ5-52, Vλ1-51,Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37,Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14,Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, or Vλ3-1 in the firstengineered endogenous immunoglobulin κ light chain locus, and whereineach of the one or more human Vλ non-coding sequences naturally appearsadjacent to a Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44,Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22,Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8,Vλ4-3, or Vλ3-1 of an endogenous human immunoglobulin λ light chainlocus; and

(ii) one or more human Jλ non-coding sequences, wherein each of the oneor more human Jλ non-coding sequences is adjacent to the Jλ1, Jλ2, Jλ3,Jλ6 or Jλ7 in the first engineered endogenous immunoglobulin κ lightchain locus, and wherein each of the one or more human Jλ non-codingsequences naturally appears adjacent to a Jλ1, Jλ2, Jλ3, Jλ6 or Jλ7 ofan endogenous human immunoglobulin λ light chain locus.

Embodiment 212

The rodent ES cell of embodiment 210, wherein the first engineeredendogenous immunoglobulin κ light chain locus comprises:

(i) one or more human Vλ non-coding sequences, wherein each of the oneor more human Vλ non-coding sequences is adjacent to the Vλ5-52, Vλ1-51,Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37,Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16,Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3 or Vλ3-1 in thefirst engineered endogenous immunoglobulin κ light chain locus, andwherein each of the one or more human Vλ non-coding sequences naturallyappears adjacent to a Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45,Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23,Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10,Vλ3-9, Vλ2-8, Vλ4-3 or Vλ3-1 of an endogenous human immunoglobulin λlight chain locus,

(ii) one or more human Jκ non-coding sequences, wherein each of the oneor more human Jκ non-coding sequences is adjacent to the Jλ1, Jλ2, Jλ3,Jλ6 or Jλ7 in the first engineered endogenous immunoglobulin κ lightchain locus, and wherein each of the one or more human Jκ non-codingsequences naturally appears adjacent to a Jκ1, Jκ2, Jκ3, Jκ4, or Jκ5 ofan endogenous human immunoglobulin κ light chain locus.

Embodiment 213

The rodent ES cell of any one of embodiments 201-212, wherein:

(i) the one or more human Vλ gene segments comprise Vλ4-69, Vλ8-61,Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45,Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23,Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9,Vλ2-8, Vλ4-3, Vλ3-1, or any combination thereof; and

(ii) the one or more human Jλ gene segments comprise Jλ1, Jλ2, Jλ3, Jλ6,Jλ7, or any combination thereof.

Embodiment 214

The rodent ES cell of embodiment 213, wherein the first engineeredendogenous immunoglobulin κ light chain locus comprises:

(i) one or more human Vλ non-coding sequences, wherein each of the oneor more human Vλ non-coding sequences is adjacent to the Vλ4-69, Vλ8-61,Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45,Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23,Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9,Vλ2-8, Vλ4-3, or Vλ3-1 in the first engineered endogenous immunoglobulinκ light chain locus, and wherein each of the one or more human Vλnon-coding sequences naturally appears adjacent to a Vλ4-69, Vλ8-61,Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45,Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23,Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9,Vλ2-8, Vλ4-3, or Vλ3-1 of an endogenous human immunoglobulin λ lightchain locus; and

(ii) one or more human Jλ non-coding sequences, wherein each of the oneor more human Jλ non-coding sequences is adjacent to the Jλ1, Jλ2, Jλ3,Jλ6 or Jλ7 in the first engineered endogenous immunoglobulin κ lightchain locus, and wherein each of the one or more human Jλ non-codingsequences naturally appears adjacent to a Jλ1, Jλ2, Jλ3, Jλ6 or Jλ7 ofan endogenous human immunoglobulin λ light chain locus.

Embodiment 215

The rodent ES cell of embodiment 213, wherein the first engineeredendogenous immunoglobulin κ light chain locus comprises:

(i) one or more human Vλ non-coding sequences, wherein each of the oneor more human Vλ non-coding sequences is adjacent to the Vλ4-69, Vλ8-61,Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45,Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23,Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10,Vλ3-9, Vλ2-8, Vλ4-3 or Vλ3-1 in the first engineered endogenousimmunoglobulin κ light chain locus, and wherein each of the one or morehuman Vλ non-coding sequences naturally appears adjacent to a Vλ4-69,Vλ8-61, Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46,Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25,Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11,Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3 or Vλ3-1 of an endogenous humanimmunoglobulin λ light chain locus; and

(ii) one or more human Jκ non-coding sequences, wherein each of the oneor more human Jκ non-coding sequences is adjacent to the Jλ1, Jλ2, Jλ3,Jλ6 or Jλ7 in the first engineered endogenous immunoglobulin κ lightchain locus, and wherein each of the one or more human Jκ non-codingsequences naturally appears adjacent to a Jκ1, Jκ2, Jκ3, Jκ4, or Jκ5 ofan endogenous human immunoglobulin κ light chain locus.

Embodiment 216

The rodent ES cell of any one of embodiments 201-215, wherein the genomeof the rodent ES cell further comprises:

an engineered endogenous immunoglobulin heavy chain locus, comprising:

-   -   (a) one or more human V_(H) gene segments,    -   (b) one or more human D_(H) gene segments, and    -   (c) one or more human J_(H) gene segments,

wherein the one or more human V_(H) gene segments, the one or more humanD_(H) gene segments, and the one or more human J_(H) gene segments areoperably linked to one or more rodent immunoglobulin heavy chainconstant region genes at the engineered endogenous immunoglobulin heavychain locus.

Embodiment 217

The rodent ES cell of embodiment 216, wherein the one or more humanV_(H) gene segments, one or more human D_(H) gene segments, and one ormore human J_(H) gene segments are in place of one or more rodent V_(H)gene segments, one or more rodent D_(H) gene segments, one or morerodent J_(H) gene segments, or a combination thereof.

Embodiment 218

The rodent ES cell of embodiment 216 or 217, wherein the one or morehuman V_(H) gene segments, one or more human D_(H) gene segments, andone or more human J_(H) gene segments replace one or more rodent V_(H)gene segments, one or more rodent D_(H) gene segments, one or morerodent J_(H) gene segments, or any combination thereof.

Embodiment 219

The rodent ES cell of any one of embodiments 216-218, wherein theengineered endogenous immunoglobulin heavy chain locus furthercomprises:

(i) one or more human V_(H) non-coding sequences, each of which isadjacent to at least one of the one or more human V_(H) gene segments,wherein each of the one or more V_(H) non-coding sequences naturallyappears adjacent to a human V_(H) gene segment in an endogenous humanimmunoglobulin heavy chain locus;

(ii) one or more human D_(H) non-coding sequences, each of which isadjacent to at least one of the one or more human D_(H) gene segments,wherein each of the one or more D_(H) non-coding sequences naturallyappears adjacent to a human D_(H) gene segment in an endogenous humanimmunoglobulin heavy chain locus;

(iii) one or more human J_(H) non-coding sequences, each of which isadjacent to at least one of the one or more human J_(H) gene segments,wherein each of the one or more J_(H) non-coding sequences naturallyappears adjacent to a human J_(H) gene segment in an endogenous humanimmunoglobulin heavy chain locus; or

(iv) any combination thereof.

Embodiment 220

The rodent ES cell of any one of embodiments 216-219, wherein the one ormore rodent immunoglobulin heavy chain constant region genes are one ormore endogenous rodent immunoglobulin heavy chain constant region genes.

Embodiment 221

The rodent ES cell of any one of embodiments 216-220, wherein:

(i) the one or more human V_(H) gene segments comprise V_(H)3-74,V_(H)3-73, V_(H)3-72, V_(H)2-70, V_(H)1-69, V_(H)3-66, V_(H)3-64,V_(H)4-61, V_(H)4-59, V_(H)1-58, V_(H)3-53, V_(H)5-51, V_(H)3-49,V_(H)3-48, V_(H)1-46, V_(H)1-45, V_(H)3-43, V_(H)4-39, V_(H)4-34,V_(H)3-33, V_(H)4-31, V_(H)3-30, V_(H)4-28, V_(H)2-26, V_(H)1-24,V_(H)3-23, V_(H)3-21, V_(H)3-20, V_(H)1-1⁸, V_(H)3-1⁵, V_(H)3-1³,V_(H)3-11, V_(H)3-9, V_(H)1-8, V_(H)3-7, V_(H)2-5, V_(H)7-4-1, V_(H)4-4,V_(H)1-3, V_(H)1-2, V_(H)6-1, or any combination thereof,

(ii) the one or more human D_(H) gene segments comprise D_(H)1-1,D_(H)2-2, D_(H)3-3, D_(H)4-4, D_(H)5-5, D_(H)6-6, D_(H)1-7, D_(H)2-8,D_(H)3-9, D_(H)3-10, D_(H)5-12, D_(H)6-13, D_(H)2-15, D_(H)3-16,D_(H)4-17, D_(H)6-19, D_(H)1-20, D_(H)2-21, D_(H)3-22, D_(H)6-25,D_(H)1-26, D_(H)7-27, or any combination thereof, and

(iii) the one or more human J_(H) gene segments comprise J_(H)1, J_(H)2,J_(H)3, J_(H)4, J_(H)5, J_(H)6, or any combination thereof.

Embodiment 222

The rodent ES cell of any one of embodiments 216-221, wherein theengineered endogenous immunoglobulin heavy chain locus lacks afunctional endogenous rodent Adam6 gene.

Embodiment 223

The rodent ES cell of any one of embodiments 216-222, wherein the genomeof the rodent further comprises one or more nucleotide sequencesencoding one or more rodent ADAM6 polypeptides, functional orthologs,functional homologs, or functional fragments thereof.

Embodiment 224

The rodent ES cell of embodiment 223, wherein the one or more nucleotidesequences encoding one or more rodent ADAM6 polypeptides, functionalorthologs, functional homologs, or functional fragments thereof areincluded on the same chromosome as the engineered endogenousimmunoglobulin heavy chain locus.

Embodiment 225

The rodent ES cell of embodiment 223 or 224, wherein the one or morenucleotide sequences encoding one or more rodent ADAM6 polypeptides,functional orthologs, functional homologs, or functional fragmentsthereof are included in the engineered endogenous immunoglobulin heavychain locus.

Embodiment 226

The rodent ES cell of any one of embodiments 223-225, wherein the one ormore nucleotide sequences encoding one or more rodent ADAM6polypeptides, functional orthologs, functional homologs, or functionalfragments thereof are in place of a human Adam6 pseudogene.

Embodiment 227

The rodent ES cell of any one of embodiments 223-226, wherein the one ormore nucleotide sequences encoding one or more rodent ADAM6polypeptides, functional orthologs, functional homologs, or functionalfragments thereof replace a human Adam6 pseudogene.

Embodiment 228

The rodent ES cell of any one of embodiments 223-227, wherein the one ormore nucleotide sequences encoding one or more rodent ADAM6polypeptides, functional orthologs, functional homologs, or functionalfragments thereof are between a first human V_(H) gene segment and asecond human V_(H) gene segment.

Embodiment 229

The rodent ES cell of embodiment 228, wherein the first human V_(H) genesegment is V_(H)1-2 and the second human V_(H) gene segment is V_(H)6-1.

Embodiment 230

The rodent ES cell of any one of embodiments 223-225, wherein the one ormore nucleotide sequences encoding one or more rodent ADAM6polypeptides, functional orthologs, functional homologs, or functionalfragments thereof are between a human V_(H) gene segment and a humanD_(H) gene segment.

Embodiment 231

The rodent ES cell of any one embodiments 216-230, wherein the rodentcell is homozygous for the engineered endogenous immunoglobulin heavychain locus.

Embodiment 232

The rodent ES cell of any one of embodiments 201-231, wherein the genomeof the rodent ES cell further comprises a nucleic acid sequence encodingan exogenous terminal deoxynucleotidyltransferase (TdT) operably linkedto a transcriptional control element.

Embodiment 233

The rodent ES cell of embodiment 232, wherein the TdT is a short isoformof TdT (TdTS).

Embodiment 234

The rodent ES cell of embodiment 232 or 233, wherein the transcriptionalcontrol element comprises a RAG1 transcriptional control element, a RAG2transcriptional control element, an immunoglobulin heavy chaintranscriptional control element, an immunoglobulin κ light chaintranscriptional control element, an immunoglobulin λ light chaintranscriptional control element, or any combination thereof.

Embodiment 235

The rodent ES cell of any one of embodiments 232-234, wherein thenucleic acid sequence encoding an exogenous TdT is at an immunoglobulinκ light chain locus, an immunoglobulin λ light chain locus, animmunoglobulin heavy chain locus, a RAG1 locus, or a RAG2 locus.

Embodiment 236

The rodent ES cell of any one of embodiments 201-235, wherein the rodentES cell is a rat ES cell or a mouse ES cell.

Embodiment 237

A method of making a fully human antibody specific against an antigencomprising the steps of:

-   -   (a) immunizing a rodent according to any one of embodiments 1-60        with the antigen;    -   (b) determining a nucleotide sequence that encodes a human heavy        chain variable domain of an antibody that specifically binds the        antigen and that was generated by the genetically modified mouse        and/or determining a nucleotide sequence that encodes a human λ        light chain variable domain of an antibody that specifically        binds the antigen and that was generated by the genetically        modified mouse; and    -   (c) expressing a fully human antibody by employing:        -   (i) the nucleotide sequence encoding a human heavy chain            variable domain of (b) operably linked to a human heavy            chain constant region gene, and/or        -   (ii) the nucleotide sequence encoding a human λ light chain            variable domain of (b) operably linked to a human light            chain constant region gene.

Embodiment 238

A method of making a fully human antibody specific against an antigencomprising the steps of:

(a) expressing in a mammalian cell a fully human antibody comprising twohuman λ light chains and two human heavy chains, wherein each human λlight chain includes a human λ light chain variable domain encoded by ahuman λ light chain variable region and each human heavy chain includesa human heavy chain variable domain encoded by a human heavy chainvariable region, wherein the nucleotide sequence of at least one humanheavy or λ light chain variable region was obtained from a rodentaccording to any one of embodiments 1-60; and

(b) obtaining the fully human antibody.

Embodiment 239

A method of making a fully human antibody specific against an antigencomprising the steps of:

-   -   (a) immunizing a rodent according to any one of embodiments        1-60;    -   (b) determining a human heavy chain variable domain sequence of        an antibody that specifically binds the antigen and that was        generated by the genetically modified mouse and/or determining        of a human λ light chain variable domain sequence of an antibody        that specifically binds the antigen and that was generated by        the genetically modified mouse; and    -   (c) expressing a fully human antibody by employing:        -   (i) the human heavy chain variable domain sequence of (b)            operably linked to a human heavy chain constant domain            sequence, and/or        -   (ii) the human λ light chain variable domain sequence of (b)            operably linked to a human light chain constant domain            sequence.

Embodiment 240

The method of embodiment 239, wherein employing the human heavy chainvariable domain sequence of (b) operably linked to a human heavy chainconstant domain sequence comprises expressing a nucleotide sequence thatencodes the human heavy chain variable domain sequence of (b) and thehuman heavy chain constant domain sequence.

Embodiment 241

The method of embodiment 239 or 240, wherein employing the human λ lightchain variable domain sequence of (b) operably linked to a human lightchain constant domain sequence comprises expressing a nucleotidesequence that encodes the human λ light chain variable domain sequenceof (b) and the human light chain constant domain sequence.

Embodiment 242

A method of making a fully human antibody specific against an antigencomprising the steps of:

(a) expressing in a mammalian cell said fully human antibody comprisingtwo human λ light chains and two human heavy chains, wherein each humanλ light chain includes a human light chain variable domain and eachhuman heavy chain includes a human heavy chain variable domain, whereinthe amino acid sequence of at least one human heavy or λ light chainvariable domain was obtained from a rodent of any one of embodiments1-60; and

(b) obtaining the fully human antibody.

Embodiment 243

A method for generating a human heavy or λ light chain variable domainsequence comprising the steps of:

(a) immunizing a rodent of any one of embodiments 1-60; and

(b) determining a human heavy or λ light chain variable domain sequenceof an antibody that specifically binds the antigen and that wasgenerated by the genetically modified mouse.

Embodiment 244

The method of embodiment 243, wherein determining a human heavy or λlight chain variable domain sequence comprises determining a nucleotidesequence that encodes the human heavy or λ light chain variable domainsequence.

Embodiment 245

A method of making a fully human heavy chain or a fully human lightchain comprising the steps of:

(a) immunizing a rodent of any one of embodiments 1-60;

(b) determining a human heavy or λ light chain variable domain sequenceof an antibody that specifically binds the antigen and that wasgenerated by the genetically modified mouse; and

(c) operably linking the human heavy or λ light chain variable domainsequence to a human heavy or light chain constant domain sequence,respectively, to form a fully human heavy chain or a fully human lightchain.

Embodiment 246

The method of embodiment 245, wherein operably linking the human heavyor λ light chain variable domain sequence to a human heavy or lightchain constant domain sequence, respectively, comprises operably linkinga nucleotide sequence encoding the human heavy or λ light chain variabledomain sequence to a nucleotide sequence encoding the human heavy orlight chain constant domain sequence.

Embodiment 247

A method for generating a human heavy or λ light chain variable regionsequence comprising the steps of:

-   -   (a) immunizing a rodent of any one of embodiments 1-60; and    -   (b) determining a human heavy or λ light chain variable region        sequence that encodes a human heavy or λ light chain variable        domain, respectively, of an antibody that specifically binds the        antigen and that was generated by the genetically modified        mouse.

Embodiment 248

A method of making a nucleotide sequence encoding a fully human heavychain or a fully human light chain comprising the steps of:

-   -   (a) immunizing a rodent of any one of embodiments 1-60;    -   (b) determining a human heavy or λ light chain variable region        sequence that encodes a human heavy or λ light chain variable        domain, respectively, of an antibody that specifically binds the        antigen and that was generated by the genetically modified        mouse; and    -   (c) operably linking the human heavy or λ light chain variable        region sequence to a human heavy or light chain constant region        gene, respectively, to form a nucleotide sequence encoding a        fully human heavy chain or a fully human light chain.

EQUIVALENTS

It is to be appreciated by those skilled in the art that variousalterations, modifications, and improvements to the present disclosurewill readily occur to those skilled in the art. Such alterations,modifications, and improvements are intended to be part of the presentdisclosure, and are intended to be within the spirit and scope of theinvention. Accordingly, the foregoing description and drawing are by wayof example only and any invention described in the present disclosure iffurther described in detail by the claims that follow.

Those skilled in the art will appreciate typical standards of deviationor error attributable to values obtained in assays or other processes asdescribed herein. The publications, websites and other referencematerials referenced herein to describe the background of the inventionand to provide additional detail regarding its practice are herebyincorporated by reference in their entireties.

The invention claimed is:
 1. A genetically modified mouse, whosegermline genome comprises: a first engineered endogenous immunoglobulinκ light chain locus comprising: (a) one or more unrearranged human Vλ,gene segments, (b) one or more unrearranged human Jλ, gene segments, and(c) a single mouse Cλ, gene, wherein the one or more unrearranged humanVλ, gene segments of (a) and the one or more unrearranged human Jλ, genesegments of (b) are in place of one or more endogenous mouse Vκ genesegments and one or more endogenous mouse Jκ gene segments; wherein theone or more unrearranged human Vλ, gene segments of (a) and the one ormore unrearranged human Jλ, gene segments of (b) are operably linked tothe single mouse Cλ, gene; wherein the single mouse Cλ, gene of (c) isoperably linked to a mouse kappa enhancer; wherein the geneticallymodified mouse lacks a mouse Cκ gene at the first engineered endogenousimmunoglobulin κ light chain locus; wherein the genetically modifiedmouse comprises a population of B cells that express antibodies, whereinthe antibodies include immunoglobulin λ, light chains that each include:(i) a human immunoglobulin λ, light chain variable domain encoded by oneof the one or more unrearranged human Vλ, gene segments of (a) or asomatically hypermuated variant thereof rearranged with one of one ormore unrearranged human Jλ, gene segments of (b) or a somaticallyhypermuated variant thereof, and (ii) a mouse Cλ, domain encoded by thesingle mouse Cλ, gene of (c), and wherein the mouse Cλ, domain of eachimmunoglobulin λ, light chain of the antibodies is expressed from thesingle mouse Cλ, gene of (c).
 2. The genetically modified mouse of claim1, wherein the genetically modified mouse is homozygous for the firstengineered endogenous immunoglobulin κ light chain locus.
 3. Thegenetically modified mouse of claim 1, wherein the genetically modifiedmouse is heterozygous for the first engineered endogenous immunoglobulinκ light chain locus.
 4. The genetically modified mouse of claim 1,wherein the germline genome of the genetically modified mouse comprisesa second engineered endogenous immunoglobulin κ light chain locuscomprising: (a) one or more human Vic gene segments, and (b) one or morehuman Jκ gene segments, wherein the one or more human Vic gene segmentsof (a) and the one or more human Jκ gene segments of (b) are operablylinked to a Cκ gene.
 5. The genetically modified mouse of claim 1,wherein the germline genome of the mouse further comprises: anengineered endogenous immunoglobulin heavy chain locus, comprising: (a)one or more human V_(H) gene segments, (b) one or more human D_(H) genesegments, and (c) one or more human J_(H) gene segments, wherein the oneor more human V_(H) gene segments of (a), the one or more human D_(H)gene segments of (b), and the one or more human J_(H) gene segments of(c) are operably linked to one or more mouse immunoglobulin heavy chainconstant region genes at the engineered endogenous immunoglobulin heavychain locus.
 6. The genetically modified mouse of claim 5, wherein theone or more human V_(H) gene segments of (a), one or more human D_(H)gene segments of (b), and one or more human J_(H) gene segments of (c)are in place of one or more mouse V_(H) gene segments, one or more mouseD_(H) gene segments, one or more mouse J_(H) gene segments, or acombination thereof.
 7. The genetically modified mouse of claim 5,wherein the engineered endogenous immunoglobulin heavy chain locusfurther comprises: (i) one or more human V_(H) non-coding sequences,each of which is adjacent to at least one of the one or more human V_(H)gene segments of (a), wherein each of the one or more V_(H) non-codingsequences naturally appears adjacent to a human V_(H) gene segment in anendogenous human immunoglobulin heavy chain locus; (ii) one or morehuman D_(H) non-coding sequences, each of which is adjacent to at leastone of the one or more human D_(H) gene segments of (b), wherein each ofthe one or more D_(H) non-coding sequences naturally appears adjacent toa human D_(H) gene segment in an endogenous human immunoglobulin heavychain locus; (iii) one or more human J_(H) non-coding sequences, each ofwhich is adjacent to at least one of the one or more human J_(H) genesegments of (c), wherein each of the one or more J_(H) non-codingsequences naturally appears adjacent to a human J_(H) gene segment in anendogenous human immunoglobulin heavy chain locus; or (iv) anycombination thereof.
 8. The genetically modified mouse of claim 5,wherein the one or more mouse immunoglobulin heavy chain constant regiongenes are one or more endogenous mouse immunoglobulin heavy chainconstant region genes.
 9. The genetically modified mouse of claim 5,wherein: (i) the one or more human V_(H) gene segments compriseV_(H)3-74, V_(H)3-73, V_(H)3-72, V_(H)2-70, V_(H)1-69, V_(H)3-66,V_(H)3-64, V_(H)4-61, V_(H)4-59, V_(H)l-58, V_(H)3-53, V_(H)5-51,V_(H)3-49, V_(H)3-48, V_(H)1-46, V_(H)1-45, V_(H)3-43, V_(H)4-39,V_(H)4-34, V_(H)3-33, V_(H)4-31, V_(H)3-30, V_(H)4-28, V_(H)2-26,V_(H)l-24, V_(H)3-23, V_(H)3-21, V_(H)3-20, V_(H)1-18, V_(H)3-15,V_(H)3-13, V_(H)3-11, V_(H)3-9, V_(H)1-8, V_(H)3-7, V_(H)2-5,V_(H)7-4-1, V_(H)4-4, V_(H)1-3, V_(H)1-2, V_(H)6-1, or a combinationthereof, (ii) the one or more human D_(H) gene segments compriseD_(H)1-1, D_(H)2-2, D_(H)3-3, D_(H)4-4, D_(H)5-5, D_(H)6-6, D_(H)1-7,D_(H)2-8, D_(H)3-9, D_(H)3-10, D_(H)5-12, D_(H)6-13, D_(H)2-15,D_(H)3-16, D_(H)4-17, D_(H)6-19, D_(H)1-20, D_(H)2-21, D_(H)3-22,D_(H)6-25, D_(H)1-26, D_(H)7-27, or a combination thereof, and (iii) theone or more human J_(H) gene segments comprise J_(H)1, J_(H)2, J_(H)3,J_(H)4, J_(H)5, J_(H)6, or a combination thereof.
 10. The geneticallymodified mouse of claim 5, wherein the mouse is homozygous for theengineered endogenous immunoglobulin heavy chain locus.
 11. Thegenetically modified mouse of claim 1, wherein the first engineeredendogenous immunoglobulin κ light chain locus further comprises a κlight chain non-coding sequence between the one or more unrearrangedhuman Vλ, gene segments and the one or more unrearranged human Jλ, genesegments.
 12. The genetically modified mouse of claim 11, wherein the κlight chain non-coding sequence has a sequence that naturally appearsbetween a human Vκ4-1 gene segment and a human Jκ1 gene segment in anendogenous human immunoglobulin κ light chain locus.
 13. The geneticallymodified mouse of claim 1, wherein endogenous Vλ, gene segments,endogenous Jλ, gene segments, and endogenous Cλ, genes are deleted inwhole or in part.
 14. The genetically modified mouse of claim 1, whereinthe germline genome of the genetically modified mouse further comprisesa nucleic acid sequence encoding an exogenous terminaldeoxynucleotidyltransferase (TdT) operably linked to a transcriptionalcontrol element.